Anti-glucosaminidase passive immunization for staphylococcus aureus infections

ABSTRACT

The present invention is directed to a monoclonal antibody that binds specifically to a Staphylococcus aureus glucosaminidase and inhibits in vivo growth of S. aureus. Also disclosed are monoclonal antibody binding portions, recombinant or hybridoma cell lines, pharmaceutical compositions containing the monoclonal antibody or binding portions thereof, and methods of treating S. aureus infection and osteomyelitis, and methods for introducing an orthopedic implant into a patient using the monoclonal antibody, binding portion, or pharmaceutical composition of the present invention.

This application is a continuation of U.S. patent application Ser. No. 15/662,715 filed on Jul. 28, 2017, which is a continuation of U.S. patent application Ser. No. 13/696,019, now U.S. Pat. No. 9,737,601, which is a national stage application under 35 U.S.C. § 371 of PCT Application No. PCT/US2011/035033, filed May 3, 2011, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/330,568, filed May 3, 2010, which is hereby incorporated by reference in its entirety.

This invention was made with government support under R43 AI085844 awarded by National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to passive immunization against Staphylococcus aureus infection, particularly for the prevention or treatment of osteomyelitis and for implantation of an orthopedic implant or graft. Antibodies that bind specifically to S. aureus glucosaminidase and pharmaceutical compositions containing the same can be used for these purposes.

BACKGROUND OF THE INVENTION

There is a great need for novel interventions of chronic osteomyelitis (OM) as approximately 112,000 orthopedic device-related infections occur per year in the US, at an approximate hospital cost of $15,000-70,000 per incident (Darouiche, “Treatment of Infections Associated With Surgical Implants,” N. Engl. J. Med. 350(14):1422-9 (2004)). Although improvements in surgical technique and aggressive antibiotic prophylaxis have decreased the infection rate following orthopedic implant surgery to 1-5%, osteomyelitis (OM) remains a serious problem and appears to be on the rise from minimally invasive surgery (Mahomed et al., “Rates and Outcomes of Primary and Revision Total Hip Replacement in the United States Medicare Population,”J. Bone Joint Surg. Am. 85(A-1):27-32 (2003); WHO Global Strategy for Containment of Antimicrobial Resistance, 2001). The significance of this resurgence, 80% of which is due to Staphylococcus aureus, is amplified by the fact that ˜50% of clinical isolates are methicillin resistant S. aureus (MRSA). While the infection rates for joint prosthesis and fracture-fixation devices have been only 0.3-11% and 5-15% of cases, respectively, over the last decade (Lew and Waldvogel, “Osteomyelitis,” Lancet 364(9431):369-79 (2004); Toms et al., “The Management of Pen-Prosthetic Infection in Total Joint Arthroplasty,” J. Bone Joint Surg. Br. 88(2):149-55 (2006)), this result may lead to amputation or death. Additionally, the popularization of “minimally invasive surgery” for elective total joint replacements (TJR) in which the very small incision often leads to complications from the prosthesis contacting skin during implantation, has markedly increased the incidence of OM (Mahomed et al., “Rates and Outcomes of Primary and Revision Total Hip Replacement in the United States Medicare Population,” J. Bone Joint Surg. Am. 85(A-1):27-32 (2003); WHO Global Strategy for Containment of Antimicrobial Resistance, 2001). These infections require a very expensive two-stage revision surgery, and recent reports suggest that success rates could be as low as 50% (Azzam et al., “Outcome of a Second Two-stage Reimplantation for Periprosthetic Knee Infection,” Clin. Orthop. Relat. Res. 467(7):1706-14 (2009)). However, the greatest concern is the emergence of drug resistant strains, most notably MRSA, which has surpassed HIV as the most deadly pathogen in North America, and continues to make the management of chronic OM more difficult, placing a great demand for novel therapeutic interventions. There is a great need for alternative interventional strategies, particularly for immune compromised elderly who are the primary recipients of TJR.

Presently, there are no prophylactic treatments that can protect high-risk patients from MRSA, most notably the aging “baby boomers” who account for most of the 1.5 million TJR performed annually in the United States. A vaccine that would decrease the MRSA incidence by 50-80% would not only reduce the number one complication of joint replacement and open fracture repair procedures, but also cut the healthcare burden by a similar amount.

Studies have documented that 80% of chronic OM is caused by S. aureus. These bacteria contain several factors that make them bone pathogens including several cell-surface adhesion molecules that facilitate their binding to bone matrix (Flock et al., “Cloning and Expression of the Gene for a Fibronectin-Binding Protein From Staphylococcus aureus,” Embo. J. 6(8):2351-7 (1987)), toxins capable of stimulating bone resorption (Nair et al., “Surface-Associated Proteins From Staphylococcus aureus Demonstrate Potent Bone Resorbing Activity,” J. Bone Miner. Res. 10(5):726-34 (1995)), through increased osteoclast activity (Marriott et al., “Osteoblasts Express the Inflammatory Cytokine Interleukin-6 in a Murine Model of Staphylococcus aureus Osteomyelitis and Infected Human Bone Tissue,” Am. J. Pathol. 164(4):1399-406 (2004)). The rate-limiting step in the evolution and persistence of infection is the formation of biofilm around implanted devices (Costerton et al., “Bacterial Biofilms: A Common Cause of Persistent Infections,” Science 284(5418):1318-22 (1999)). Shortly after implantation, a conditioning layer composed of host-derived adhesins (including fibrinogen, fibronectin, and collagen) forms on the surface of the implant and invites the adherence of free-floating bacteria derived from hematogenous seeding, including spread of infection from a contiguous area (the skin adjacent to a wound), surgical inoculation of bacteria into bone, or trauma coincident with significant disruption of the associated soft tissue bone envelope (Darouiche, “Treatment of Infections Associated With Surgical Implants,” N. Engl. J. Med. 350(14):1422-9 (2004)). Over the next few days bacterial cell division, recruitment of additional planktonic organisms, and secretion of bacterial products (such as the glycocalyx) produces the biofilm. This biofilm serves as a dominant barrier to protect the bacteria from the action of antibiotics, phagocytic cells, antibodies and impairs lymphocyte functions (Gray et al., “Effect of Extracellular Slime Substance From Staphylococcus epidermidis on the Human Cellular Immune Response,” Lancet 1(8373):365-7 (1984); Johnson et al., “Interference With Granulocyte Function By Staphylococcus epidermidis Slime,” Infect. Immun. 54(1):13-20 (1986); Naylor et al., “Antibiotic Resistance of Biomaterial-Adherent Coagulase-Negative and Coagulase-Positive Staphylococci,” Clin. Orthop. Relat. Res. 261:126-33 (1990)).

Another recent discovery is that S. aureus not only colonizes bone matrix, but is also internalized by osteoblasts in vitro (Ellington et al., “Involvement of Mitogen-Activated Protein Kinase Pathways in Staphylococcus aureus Invasion of Normal Osteoblasts,” Infect. Immun. 69(9):5235-42 (2001)) and in vivo (Reilly et al., “In Vivo Internalization of Staphylococcus aureus by Embryonic Chick Osteoblasts,” Bone 26(1):63-70 (2000)). This provides yet another layer of antibody and antibiotic resistance. This phase of infection occurs under conditions of markedly reduced metabolic activity and sometimes appears as so-called small-colony variants that likely accounts for its persistence (Proctor et al., “Persistent and Relapsing Infections Associated with Small-Colony Variants of Staphylococcus aureus,” Clin. Infect. Dis. 20(1):95-102 (1995)). At this point the bacteria may also express phenotypic resistance to antimicrobial treatment, also explaining the high failure rate of short courses of therapy (Chuard et al., “Resistance of Staphylococcus aureus Recovered From Infected Foreign Body in Vivo to Killing by Antimicrobials,” J. Infect. Dis. 163(6):1369-73 (1991)). Due to these extensive pathogenic mechanism, OM is notorious for its tendency to recur even after years of quiescence, and it is accepted that a complete cure is an unlikely outcome (Mader and Calhoun, “Long-Bone Osteomyelitis Diagnosis and Management,” Hosp. Pract. (Off Ed) 29(10):71-6, 9, 83 passim (1994)).

One of the key questions in the field of chronic OM is why current knowledge of factors that regulate chronic OM so limited. Supposedly, the experimental tools necessary to elucidate bacterial virulence gene have been available for over a century. There are three explanations for this anomaly. First, although the total number of osteomyelitis cases is high, its incidence of 1-5% is too low for rigorous prospective clinical studies, with the possible exception of revision arthropasty. Second, it is well known that in vitro cultures rapidly select for growth of organisms that do not elaborate an extracellular capsule, such that biofilm biology can only be studied with in vivo models (Costerton et al., “Bacterial Biofilms: A Common Cause of Persistent Infections,” Science 284(5418):1318-22 (1999)). This leads to the “greatest obstacle” in this field, which is the absence of a quantitative animal model that can assess the initial planktonic growth phase of the bacteria prior to biofilm formation. To date, much of the knowledge of its pathogenesis comes from animal models (Norden, “Lessons Learned From Animal Models of Osteomyelitis,” Rev. Infect. Dis. 10(1):103-10 (1988)), which have been developed for the chicken (Daum et al., “A Model of Staphylococcus aureus Bacteremia, Septic Arthritis, and Osteomyelitis in Chickens,” J. Orthop. Res. 8(6):804-13 (1990)), rat (Rissing et al., “Model of Experimental Chronic Osteomyelitis in Rats,” Infect. Immun. 47(3):581-6 (1985)), guinea pig (Passl et al., “A Model of Experimental Post-Traumatic Osteomyelitis in Guinea Pigs,” J. Trauma 24(4):323-6 (1984)), rabbit (Worlock et al., “An Experimental Model of Post-Traumatic Osteomyelitis in Rabbits,” Br. J. Exp. Pathol. 69(2):235-44 (1988)), dog (Varshney et al., “Experimental Model of Staphylococcal Osteomyelitis in Dogs,” Indian J. Exp. Biol. 27(9):816-9 (1989)), sheep (Kaarsemaker et al., “New Model for Chronic Osteomyelitis With Staphylococcus aureus in Sheep,” Clin. Orthop. Relat. Res. 339:246-52 (1997)) and most recently mouse (Marriott et al., “Osteoblasts Express the Inflammatory Cytokine Interleukin-6 in a Murine Model of Staphylococcus aureus Osteomyelitis and Infected Human Bone Tissue,” Am. J. Pathol. 164(4):1399-406 (2004)). While these models have been used to confirm the importance of bacterial adhesions identified from in vitro assays (Chuard et al., “Susceptibility of Staphylococcus aureus Growing on Fibronectin-Coated Surfaces to Bactericidal Antibiotics,”Antimicrob. Agents Chemother. 37(4):625-32 (1993); Buxton et al., “Binding of a Staphylococcus aureus Bone Pathogen to Type I Collagen,”Microb. Pathog. 8(6):441-8 (1990); Switalski et al., “A Collagen Receptor on Staphylococcus aureus Strains Isolated From Patients With Septic Arthritis Mediates Adhesion to Cartilage,”Mol. Microbiol. 7(1):99-107 (1993)), they do not have an outcome measure of in vivo growth, bacterial load, or osteolysis. Thus, they cannot be efficiently used to assess drug effects, bacterial mutants, and the role of host factors with transgenic mice.

Based on over 150 years of research, a clear paradigm to explain microbial pathogenesis has emerged. This model also applies to OM. The initial step of infection occurs when a unicellular bacterium invades the body. At this point the microbe must respond to environmental changes and express virulence genes that will help it defeat innate immunity and provide it with adhesin receptors to attach to the host. The bacterium is also dependent on the stochastic availability of host adhesins from necrotic tissue or a foreign body such as an implant. Successful completion of these steps leads to an exponential growth phase, which ceases at the point of nutrient exhaustion and/or the development of adaptive immunity. Following the exponential growth phase the bacteria are forced to persist under dormant growth conditions within the biofilm. However, at this point the infection is now chronic and cannot be eradicated by drugs or host immunity. Thus, the focus in this field has been on cell surface adhesins that specifically interact with extracellular matrix components known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) (Patti et al., “MSCRAMM-Mediated Adherence of Microorganisms to Host Tissues,”Annu. Rev. Microbiol. 48:585-617 (1994)). In fact, essentially all anti-S. aureus vaccines that have been developed to date have been directed against MSCRAMMs that are important for host tissue colonization and invasion. The goal of these vaccines is to generate antibodies that bind to these surface antigens, thereby inhibiting their attachment to host tissue. By opsinizing the bacterial surface, these antibodies can also mediate S. aureus clearance by phagocytic cells. Unfortunately, S. aureus has many adhesins, such that inhibition of one or more may not be sufficient to prevent bacterial attachment. Furthermore, bacterial clearance by phagocytic cells may be limited in avascular tissue, such that mAb may need additional anti-microbial mechanism of action to significantly reduce the in vivo planktonic growth of S. aureus and prevent the establishment of chronic OM or reinfection during revision total joint replacement surgery.

The present invention is directed to overcoming these and other deficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a monoclonal antibody or binding portion thereof that binds specifically to a Staphylococcus aureus glucosaminidase and inhibits in vivo growth of S. aureus. In one embodiment, the monoclonal antibody or binding portion thereof binds specifically to an epitope lying wholly or partly within an R3 domain of the S. aureus glucosaminidase.

A second aspect of present invention relates to a cell line that expresses a monoclonal antibody or binding portion of the present invention. In one embodiment, the cell line is a hybridoma cell line. In another embodiment, the cell line is a recombinant cell line that expresses the antibody.

A third aspect of the present invention relates to a pharmaceutical composition that may include a carrier and one or more monoclonal antibodies or binding portions of the present invention.

A fourth aspect of the present invention relates to a method of treating S. aureus infection that may include administering to a patient having a S. aureus infection an effective amount of a monoclonal antibody, binding portion, or pharmaceutical composition of the present invention.

A fifth aspect of the present invention relates to a method of treating osteomyelitis that may include administering to a patient having a S. aureus bone or joint infection an effective amount of a monoclonal antibody, binding portion, or pharmaceutical composition of the present invention.

A sixth aspect of the present invention relates to a method of introducing an orthopedic implant into a patient that may include administering to a patient in need of an orthopedic implant an effective amount of a monoclonal antibody, binding portion, or pharmaceutical composition of the present invention, and introducing the orthopedic implant into the patient. In this aspect of the present invention, the monoclonal antibody, binding portion, or pharmaceutical composition acts as a prophylactic agent. In certain embodiments, this aspect of the invention is directed to preventing OM or S. aureus reinfection during or subsequent to revision total joint replacement surgery.

Because S. aureus, and especially antibiotic resistant variants such as methicillin resistant S. aureus (MRSA), are the most common and challenging causes of Staphylococcus infections, the methods of the present invention aim to disrupt critical steps in the growth cycle of these microorganisms. The present invention also relates to a passive immunization for preventing infections in patients, for example, patients undergoing total joint replacement. The selected target for immunization is the glucosaminidase (Gmd) that S. aureus secretes to facilitate cytokinesis, the separation of cells during mitosis (Oshida et al., “A Staphylococcus aureus Autolysin that has an N-acetylmuramoyl-L-Alanine Amidase Domain and an Endo-beta-N-acetylglucosaminidase Domain: Cloning, Sequence Analysis, and Characterization,” Proc Natl Acad Sci USA 92:285-9 (1995); Oshida et al., “Expression Analysis of the Autolysin Gene (ad) of Staphylococcus aureus,” Microbiol Immunol 42:655-9 (1998); Sugai et al., “Localized Perforation of the Cell Wall by a Major Autolysin: atl Gene Products and the Onset of Penicillin-induced Lysis of Staphylococcus aureus,” J Bacteriol 179:2958-62 (1997); and Yamada et al., “An Autolysin Ring Associated with Cell Separation of Staphylococcus aureus,” J Bacteriol 178:1565-71 (1996), which are hereby incorporated by reference in their entirety).

To study and evaluate S. aureus infections, OM and various therapies directed towards Staphylococcus infections, a novel murine model of implant-associated OM in which a stainless steel pin is coated with S. aureus and implanted transcortically through the tibial metaphysic was used (Li et al., “Quantitative Mouse Model of Implant-Associated Osteomyelitis and the Kinetics of Microbial Growth, Osteolysis, and Humoral Immunity,” J Orthop. Res. 26(1):96-105 (2008), which is hereby incorporated by reference in its entirety). This model provides highly reproducible OM with Gram-positive biofilm, osteolysis, sequestrum/involucrum formation, and closely resembles clinical OM. Furthermore, in vivo bioluminescence imaging was used to quantify the planktonic growth phase of the bacteria; real time quantitative-PCR (RTQ-PCR) was used to determine nuc gene copy number in infected bone tissue to quantify the total bacteria load; and micro-CT was used to quantify osteolysis.

Using the above-mentioned murine model of osteomyelitis, antibodies specific for Gmd have been identified as a conspicuous part of the successful immune response in the challenged mice. In addition, a vaccine comprising recombinant Gmd with N-terminal His₆ (Gmd-His) elicited at least partial immunity in the mouse model. The anti-Gmd antibodies can block S. aureus cell division by either directly blocking cell division or by recruiting host effectors such as phagocytes or complement at a vulnerable point in the cycle of cell division.

Experiments demonstrating the action of individual monoclonal antibodies on the cell growth of S. aureus are presented in detail in the accompanying examples. The specific objective was to determine if single antibodies, in the absence of any immune effectors, would suppress or alter the growth of rapidly dividing S. aureus. The growth-related increase in light-scattering by growing cultures of S. aureus Xen29 was reduced by five selected monoclonal antibodies, but they did not appear to actually alter the in vitro growth rate per se. Rather, they appear to have reduced the activity of Gmd to a degree such that dividing cells failed to separate from each other. The effect was dose-dependent and consistent with a high affinity interaction between each antibody and Gmd. These effects demonstrate that these antibodies, raised against recombinant Gmd, react effectively with native Gmd and diminish its enzymatic activity. One of the monoclonal antibodies, 1C11, demonstrated the unique ability to promote cell-independent lysis of S. aureus, and two monoclonal antibodies, 1C11 and 3A8, demonstrated an ability to inhibit in vivo S. aureus growth and infection during orthopedic implant surgery in an in vivo mouse model.

Accordingly, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the quantification of osteolysis from implant-associated osteomyelitis. A longitudinal series of X-rays from a representative mouse demonstrate the development of implant-associated osteolysis over time in this model (FIG. 1A). Medial views of reconstructed μCT (micro-computed tomography) images of representative tibiae from mice (N=5) that received a trans-tibial pin coated with S. aureus and were sacrificed on the indicated day (FIG. 1B). Also shown are control mice that received a trans-tibial pin coated with S. aureus and treated with parenteral gentamicin (Gent), or received a sterile pin. The osteolytic area around the pin was quantified as previously described (Li et al., “Quantitative Mouse Model of Implant-Associated Osteomyelitis and the Kinetics of Microbial Growth, Osteolysis, and Humoral Immunity,” J. Orthop. Res. 26(1):96-105 (2008), which is hereby incorporated by reference in its entirety), and the data are presented as the mean+/−SD (* p<0.05 vs. Day 4; ** p<0.05 vs. Gent Day 18) (FIG. 1C). There was no difference in the osteolysis area between the gentamicin and sterile pin controls.

FIGS. 2A-H show the histology of trans-tibial implant-associated OM. H&E (Haematoxylin and Eosin stain) (FIGS. 2A-C), TRAP (Tartrate-Resistant Acid Phosphatase) (FIGS. 2D-F) and Gram stained (FIGS. 2G and 2H) sections of histology at the pin site (*) adjacent to the tibial cortex (#), 9 days after implantation of a sterile pin (FIGS. 2A, 2D, and 2G), or a pin coated with S. aureus (FIGS. 2B, 2C, 2E, 2F, and 2H). Of note is the new bone (h) that forms around the sterile pin (FIGS. 2A, 2D, and 2G) vs. the necrotic sequestrum (s) and involucrum (i) adjacent to the infected pin. While very few TRAP+ osteoclasts (yellow arrow heads) were present in the uninfected samples (FIG. 2D), numerous osteoclasts appear to be actively resorbing the cortex adjacent to the infected pin, and remodeling the new woven bone that is encasing the involucrum (FIGS. 2E and 2F). Gram staining confirmed the absence of bacteria in the specimens with the sterile pin (FIG. 2G) and their presence (black arrow heads) within the necrotic bone around the infected pins.

FIGS. 3A-C show the inverse correlation between bacterial load and humoral immunity against S. aureus antigens during the establishment of chronic osteomyelitis. A time course study was performed in which mice were given an infected transcortical pin containing 1×10⁶ CFU of S. aureus in their tibia and sacrificed on the indicated day. At sacrifice, DNA was purified from the infected tibia and RTQ-PCR was performed to determine the Ct values for S. aureus nuc. Using a standard curve shown, this number was converted to the recoverable nuc genes per tibia. To control for the integrity of the samples, the recoverable nuc gene per tibia value was standardized to the Ct value for mouse β-actin for each sample. From this conversion the bacterial load was derived as “Nuc Gene Copies/Tibia.” The data from each mouse is shown in FIG. 3A as an individual point, and the mean+/−SD for each time point (n=5) is presented in FIG. 3B. To assess the development of anti-S. aureus specific antibodies during the establishment of OM, serum was taken from each mouse in the group that was sacrificed on day 18, before infection (day 0) and on days 4, 7, 11, 14 and 18 after infection. This serum was used as the primary antibody in Western blots of total S. aureus extract that were then probed with HRP-conjugated antibodies that are specific for mouse IgG as shown in FIG. 3C. The data show that there is a steady increase in bacterial growth from day 0 to day 11, when the host first develops specific antibodies against the bacteria. As the titer of the anti-S. aureus antibodies increases the bacterial load drops, suggesting that the antibodies are protective. The Western blots also clearly identify four immuno-dominant antigens of 26, 34, 38 and 56 kDa (arrows). It has also been demonstrated that Xen 29 also induces antibodies against these same 26, 34, 38 and 56 kDa proteins.

FIGS. 4A-C show that glucosaminidase of S. aureus autolysin is the 56 kDa immuno-dominant antigen. To elucidate the molecular identity of the novel S. aureus antigens identified in FIG. 3, subtractive immunoblot analysis of 2D-SDS-PAGE of whole cell extract was performed with pre-immune and day 14 immune sera. Three 2D gels were run after isoelectric focusing (pH 4.0-10.0). The first was Coomassie blue-stained (FIG. 4A). The others were Western blotted with either day 0 (FIG. 4B) or day 14 sera (FIG. 4C). In addition to the background reactivity, the immune serum detected a specific polypeptide (˜53 kDa; pH 9: arrow). The 53 kDa spot was removed from the Coomassie gel, digested with trypsin, and analyzed by MALDI, which resolved 70 individual peptide peaks. The amino acid sequence from every peptide was a 100% match with the known sequence of the glucosaminidase of S. aureus autolysin, which is 53.6 kDa and has a pI of 9.66.

FIGS. 5A-B show bioluminescent imaging (BLI) quantification of bacterial growth during the establishment of chronic osteomyelitis. FIG. 5A shows BLI levels (p/sec/cm²/sr) at the site of infection and was assessed longitudinally in mice that received a sterile trans-tibial pin (Uninfected), or a pin coated with Xen 29 S. aureus (Infected) and were imaged on the indicated day. The circle in the top left image highlights the 1.5 cm diameter region of interest (ROI) that was assessed for BLI in each mouse at each time point. FIG. 5B shows the data from mice (N=5) that were Uninfected, Infected or infected and treated with parenteral antibiotics (Gentamycin) and were assessed for BLI longitudinally at the indicated time following surgery. The data are presented as the mean+/−SD (* Significantly greater vs. Day 0; p<0.05).

FIGS. 6A-B show that functional anti-Gmd ELISA demonstrated the efficacy of recombinant Gmd vaccine. FIG. 6A shows serum ELISA in which His-Gmd was used as the antigen to assay anti-Gmd antibody titers in mouse serum which was generated using a known high titer anti-sera from S. aureus infected mice. The serial dilution factor (X axis) and absorbance reading at 450 nm (Y axis) of the serial 2-fold diluted sera samples are plotted in the XY plane using GraphPad Prism 4 software. The functional titer (1:3623) is extrapolated from the inflection point (arrow) of the dilution curve. FIG. 6B shows the ELISA used to determine the titers of anti-Gmd antibodies in the sera of mice pre-immunization, pre-boost and pre-challenge with the indicated vaccine. Note that only mice immunized with the His-Gmd vaccine obtained high titers.

FIGS. 7A-C show that recombinant His-Gmd vaccine protects mice from implant-associated OM. The mice (n=20) were challenged with a Xen29 infected transtibial pin as described in the accompanying Examples, BLI was performed on day 3, and the mice were euthanized for nuc RTQ-PCR on day 11. An image of the BLI from a representative mouse in Group 1 & 3 is shown (FIG. 7A), and the mean+/−SD is presented to show the significant reduction BLI (FIG. 7B). This translated into a significant decrease in amplifiable nuc genes (mean+/−SD) on day 11 (FIG. 7C).

FIG. 8 shows the in vitro growth of S. aureus Xen29 in the presence of anti-Gmd monoclonal antibodies (anti-Gmd mAb). 100 cfu of Xen29 from a culture in log-phase growth were incubated at 37° C. with anti-Gmd monoclonal antibodies 1C11, 1E12, 2D11, and 3A8, 50 μg/mL in LB medium. Growth was monitored by light scattering at both 670 and 490 nm at the indicated intervals. MOPC21 is the isotype-matched control antibody.

FIG. 9 shows the dose-dependent effect of anti-Gmd mAb 1C11 on in vitro S. aureus growth. 100 cfu of Xen29 from a culture in log-phase growth were incubated at 37° C. with a range of concentrations of anti-Gmd mAb 1C11 in LB medium. Growth was monitored by light scattering at both 670 and 490 nm at the indicated intervals.

FIG. 10 shows the effect of Control mAb MOPC21 on in vitro S. aureus growth. 100 cfu of Xen29 from a culture in log-phase growth were incubated at 37° C. with a range of concentrations of isotype-matched control monoclonal antibody MOPC21 in LB medium. Growth was monitored by light scattering at both 670 and 490 nm at the indicated intervals. Note that the slight elevation of the 50 μg/mL line is due to the use of outside wells on the microtiter plate where temperatures equilibrate faster.

FIG. 11 shows a ClustalW amino acid sequence alignment of the V_(H) sequences from hybridomas 2D11, 3H6, 1E12 and 3A8. (2D11 V_(H)=SEQ ID NO: 1; 3H6 V_(H)=SEQ ID NO: 2; 1E12 V_(H)=SEQ ID NO: 3; 3A8 V_(H)=SEQ ID NO: 4). Highlighted sequences indicate the putative complementarity determining regions (CDR) in 2D11. A consensus sequence (SEQ ID NO: 31) is derived from these hybridoma sequences.

FIG. 12 shows a ClustalW amino acid sequence alignment of V_(t), sequences from hybridomas 1E12 and 2D11 (1E12 V_(L)=SEQ ID NO: 10; 2D11 V_(L)=SEQ ID NO: 8). A consensus sequence (SEQ ID NO: 32) is derived from these two hybridoma sequences.

FIG. 13 shows a ClustalW amino acid sequence alignment of the V_(H) sequences from hybridomas 2D11, 3H6, 1E12, 3A8, and 1C11 (2D11 V_(H)=SEQ ID NO: 1; 3H6 V_(H)=SEQ ID NO: 2; 1E12 V_(H)=SEQ ID NO: 3; 3A8 V_(H)=SEQ ID NO: 4; 1C11 V_(H)=SEQ ID NO: 5). A consensus sequence (SEQ ID NO: 6) is derived from these five hybridoma sequences.

FIGS. 14A-B show a ClustalW amino acid sequence alignment of the V_(L) sequences. FIG. 14A shows V_(L) alignment of hybridomas 1E12, 2D11, 3A8, 3H6, and 1C11 (1E12 V_(L)=SEQ ID NO: 10; 2D11 V_(L)=SEQ ID NO: 8; 3A8 V_(L)=SEQ ID NO: 11; 3H6 V_(L)=SEQ ID NO: 9; 1C11 V_(L)=SEQ ID NO: 12). A consensus sequence (SEQ ID NO: 13) is derived from these five hybridoma sequences. FIG. 14B shows V_(L) alignment of hybridomas 1E12, 2D11, 3A8, and 3H6 (1E12 V_(L)=SEQ ID NO: 10; 2D11 V_(L)=SEQ ID NO: 8); 3A8 V_(L)=SEQ ID NO: 11; 3H6 V_(L)=SEQ ID NO: 9). A consensus sequence (SEQ ID NO: 33) is derived from these four hybridoma sequences.

FIG. 15 shows a ClustalW amino sequence alignment of the V_(H) sequences from hybridomas 3A8 and 1C11 (3A8 V_(H)=SEQ ID NO: 4; 1C11 V_(H)=SEQ ID NO: 5). A consensus sequence (SEQ ID NO: 7) is derived from these two hybridoma sequences.

FIG. 16 shows a ClustalW amino acid sequence alignment of the V_(L) sequences from hybridomas 3A8 and 1C11 (3A8 V_(L)=SEQ ID NO: 11; 1C11 V_(L)=SEQ ID NO: 12). A consensus sequence (SEQ ID NO: 14) is derived from these two hybridoma sequences.

FIG. 17A illustrates an alignment of the 1C11 V_(H) domain (SEQ ID NO: 5) with a homologous amino acid sequence (SEQ ID NO: 19) encoded by the human gene IGV7-81 (see Genbank Accession AAH32733 and BC032733, each of which is hereby incorporated by reference in its entirety). A consensus sequence for the V_(H) homologs (SEQ ID NO: 34) is shown. FIG. 17B illustrates an alignment of the 1C11 V_(L) domain (SEQ ID NO: 12) with a homologous amino acid sequence (SEQ ID NO: 20) encoded by the human gene IGVK6D-21 (see Genbank Accession AAA58917 and M29469, each of which is hereby incorporated by reference in its entirety). A consensus sequence for the V_(L) homologs (SEQ ID NO: 35) is shown.

FIG. 18 shows the inhibition of S. aureus His-Gmd (Gmd) and hen egg lysozyme (HEL) by the five anti-Gmd monoclonal antibodies with MOPC21 as an isotype-matched negative control. The concentration of the antibody in μg/mL is listed on the x-axis; the inhibition of enzyme activity in percentage (%) is listed on the Y-axis. All five anti-Gmd mAbs (1C11, 1E12, 2D11, 3A8, and 3H6) inhibit Gmd activity, but have no effect on HEL activity, and MOPC21 (negative control) does not inhibit either enzyme.

FIG. 19 shows inhibition of native Gmd by the five anti-His-Gmd mAbs 1C11, 1E12, 2D11, 3A8, and 3H6. Each antibody was added at a concentration of 100 μg/mL. All five are potent inhibitors and inhibit the native enzyme to about the same degree as they inhibit the recombinant Gmd-His. The isotype-matched (IgG1) antibody control MOPC21 had no effect on Gmd enzymatic activity.

FIGS. 20A-D are scanning electron miscroscopy (SEM) images of S. aureus grown in the absence (FIGS. 20A-B) or presence of anti-Gmd monoclonal antibodies of the present invention. FIG. 20C shows the effects of 50 μg/ml mAb 1E12 on Xen29 S. aureus and FIG. 20D shows the effects of 50 ng/ml mAb 1C11 on Xen29 S. aureus. Micrographs of representative fields were obtained at 50,000× (A&C), 2,000× (B) and 4,000× (D). Arrows identify sites where lysis has occurred, and document the surprising and unexpected effects of the present invention, as complement and immune effector cell independent lytic activity of an anti-S. aureus mAb has yet to be documented in the literature.

FIGS. 21A-C show that passive immunization with monoclonal antibody 3A8 inhibits S. aureus growth in vivo and protects mice from implant-associated osteomyelitis. The mice were imaged to assess bioluminescence on days 0, 3, 5, 7, 11, and 14, and images with the BLI heat map from a representative animal in each group are shown in FIG. 21A. The BLI values on day 3 for each mouse in the study are shown with the mean for each group (FIG. 21B, p=0.02). X-rays from a representative animal in each group obtained on day 14 are shown to illustrate the osteolytic lesion (arrow) in the placebo mouse, which was not present in the anti-Gmd treated animals (FIG. 21C).

FIGS. 22A-C show that passive immunization with monoclonal antibody 1C11 inhibits S. aureus growth in vivo and protects mice from implant-associated osteomyelitis. The mice were imaged to assess bioluminescence on days 0, 3, 5, 7, 10, and 14, and images with the BLI heat map from a representative animal in each group are shown in (FIG. 22A). The BLI values on day 3 for each mouse in the study are shown with the mean for each group (FIG. 22B). X-rays from a representative animal in each group obtained on day 14 are shown to illustrate the osteolytic lesion (arrow) in the placebo mouse, which was not present in the anti-Gmd treated mouse (FIG. 22C).

FIG. 23 is a graph comparing the anti-Gmd inhibitory activity of mouse monoclonal 1C11 with the humanized chimeric monoclonal derived from 1C11. The human: mouse chimeric IgG1 of 1C11 (h1C11) retains the ability to inhibit His-Gmd. The percent inhibition of His-Gmd activity on the Y-axis is displayed as a function of dilution of the antibody preparation on the X-axis. The mouse 1C11 concentration was 10 μg/mL; the concentration for chimeric h1C11 was not known for the assay shown.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to a monoclonal antibody that binds specifically to a Staphylococcus aureus glucosaminidase and inhibits in vivo growth of S. aureus. The monoclonal antibody of the present invention can be such that it targets S. aureus that is methicillin resistant.

As used herein, the term “antibody” is meant to include immunoglobulins derived from natural sources or from recombinant sources, as well as immunoreactive portions (i.e. antigen binding portions) of immunoglobulins. The monoclonal antibodies of the present invention may exist in or can be isolated in a variety of forms including, for example, substantially pure monoclonal antibodies, antibody fragments or binding portions, chimeric antibodies, and humanized antibodies (Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 1999), which is hereby incorporated by reference in its entirety).

The monoclonal antibodies of the present invention are characterized by specificity for binding to S. aureus glucosaminidase or fragments thereof. The antibody specifically binds to an immuno-dominant epitope in the glucosaminidase (Gmd) sub-unit of S. aureus autolysin (Atl). These monoclonal antibodies inhibit in vivo growth of S. aureus.

Immuno-dominant antigen is a part of the antigenic determinant that is most easily recognized by the immune system and thus exerts the most influence on the specificity of the induced antibody. An “immuno-dominant epitope” refers to the epitope on an antigen that selectively provokes an immune response in a host organism to the substantial exclusion of other epitopes on that antigen.

Usually, the antigen likely to carry an immuno-dominant epitope can be identified by selecting antigens on the outer surface of the pathogenic organism. For example, most simple organisms, such as fungi, bacteria and viruses have one or two proteins that are exposed on the outer surface of the pathogenic organism. These outer surface proteins are most likely to carry the appropriate antigen. The proteins most likely to carry an immuno-dominant epitope can be identified in a Western assay in which total protein is run on a gel against serum from an organism infected with the pathogenic organism. Bound antibodies from the serum are identified by labeled anti-antibodies, such as in one of the well-known ELISA techniques. The immuno-dominant epitope can be identified by examining serum from a host organism infected with the pathogenic organism. The serum is evaluated for its content of antibodies that bind to the identified antigens that are likely to cause an immune response in a host organism. If an immuno-dominant epitope is present in these antigens, substantially all antibodies in the serum will bind to the immuno-dominant epitope, with little binding to other epitopes present in the antigen.

Atl is one of the catalytically distinct peptidoglycan hydrolases in S. aureus that is required to digest the cell wall during mitosis (Baba and Schneewind, “Targeting of Muralytic Enzymes to the Cell Division Site of Gram-Positive Bacteria: Repeat Domains Direct Autolysin to the Equatorial Surface Ring of Staphylococcus aureus,” EMBO. J. 17(16):4639-46 (1998), which is hereby incorporated by reference in its entirety). In addition to being an essential gene for growth, scanning electron microscopy studies have demonstrated that anti Atl antibodies bound to S. aureus during binary fission localize to regions of the bacteria that are not covered by the cell wall (Yamada et al., “An Autolysin Ring Associated With Cell Separation of Staphylococcus aureus,” J. Bacteriol. 178(6):1565-71 (1996), which is hereby incorporated by reference in its entirety).

The Atl enzyme is comprised of an amidase (62 kD) and glucosaminidase (53 kD), which are produced from the same Atl precursor protein via a cleavage process (Baba and Schneewind, “Targeting of Muralytic Enzymes to the Cell Division Site of Gram-Positive Bacteria: Repeat Domains Direct Autolysin to the Equatorial Surface Ring of Staphylococcus aureus,” Embo. J. 17(16):4639-46 (1998); Komatsuzawa et al., “Subcellular Localization of the Major Autolysin, ATL and Its Processed Proteins in Staphylococcus aureus,” Microbiol Immunol. 41:469-79 (1997); Oshida et al., “A Staphylococcus aureus Autolysin That Has an N-acetylmuramoyl-L-alanine Amidase Domain and an Endo-beta-N-acetylglucosaminidase Domain: Cloning, Sequence Analysis, and Characterization,” Proc. Nat'l. Acad. Sci. U.S.A. 92(1):285-9 (1995), which are hereby incorporated by reference in their entirety). The autolysin is held to the cell wall by three ˜150 amino acid cell wall binding domains R1, R2, and R3. In the final maturation step, proteolytic cleavage separates the aminidase domain and its associated R1 and R2 domains from the glucosaminidase and its associated N-terminal R3 domain.

By way of example, and without limitation, one exemplary Staphylococcus aureus glucosaminidase contains the amino acid sequence of SEQ ID NO: 36 below.

AYTVTKPQTT QTVSKIAQVK PNNTGIRASV YEKTAKNGAK YADRTFYVTK ERAHGNETYV LLNNTSHNIP LGWFNVKDLN VQNLGKEVKT TQKYTVNKSN NGLSMVPWGT KNQVILTGNN IAQGTFNATK QVSVGKDVYL YGTINNRTGW VNAKDLTAPT AVKPTTSAAK DYNYTYVIKN GNGYYYVTPN SDTAKYSLKA FNEQPFAVVK EQVINGQTWY YGKLSNGKLA WIKSTDLAKE LIKYNQTGMT LNQVAQIQAG LQYKPQVQRV PGKWTDANFN DVKHAMDTKR LAQDPALKYQ FLRLDQPQNI SIDKINQFLK GKGVLENQGA AFNKAAQMYG INEVYLISHA LLETGNGTSQ LAKGADVVNN KVVTNSNTKY HNVFGIAAYD NDPLREGIKY AKQAGWDTVS KAIVGGAKFI GNSYVKAGQN TLYKMRWNPA HPGTHQYATD VDWANINAKI IKGYYDKIGE VGKYFDIPQY In SEQ ID NO: 36, underlined residues correspond to residues 783 to 931 of the encoded autolysin, and represent the R3 domain. The remaining C-terminal residues (not underlined) correspond to the catalytic glucosaminidase domain.

In certain embodiments the monoclonal antibody of the present invention binds to a conserved epitope of Staphylococcus aureus glucosaminidase with an affinity greater than 10⁻⁹ M. As used herein, “epitope” refers to the antigenic determinant of Staphylococcus aureus glucosaminidase that is recognized by the monoclonal antibody. The epitope recognized by the antibody of the present invention may be a linear epitope, i.e. the primary structure of the amino acid sequence of glucosaminidase. Alternatively, the epitope recognized by the antibody of the present invention may be a non-linear or conformational epitope, i.e. the tertiary structure of glucosaminidase.

In certain embodiments, the monoclonal antibodies may bind specifically to the catalytic domain of the Gmd. In other embodiments, the monoclonal antibodies may bind specifically to the R3 domain.

Epitopes that are bound by five of the monoclonal antibodies identified herein lie wholly or at least partially within the R3 domain. By way of example, an epitope bound by mAb 3A8 lies within the region containing residues 776-842; an epitope bound by mAb 1C11 lies within the region containing residues 842-873; an epitope(s) bound by mAbs 2D11 and 1E12 are the same or different and lie within the region containing residues 842-948; and an epitope bound by mAb 3H6 lies within the region containing residues 907-948.

In certain embodiments, the monoclonal antibody of the present invention possesses S. aureus Gmd inhibitory activity, whereby the monoclonal antibody inhibits the activity of Gmd by at least 20%, at least 30%, at least 40% or at least 50%. In other embodiments, the monoclonal antibody inhibits the activity of Gmd by at least 60%, at least 70%, or at least 80%. Five monoclonal antibodies described herein (mAbs 3A8, 1C11, 2D11, 1E12, and 3H6) possess anti-Gmd inhibitory activity of about 70 to about 80 percent. It is a surprising and unexpected result that the antibodies of the present invention would bind a purported cell wall binding domain, the R3 domain, rather than a catalytic domain to inhibit enzymatic activity. Without being bound by theory, it is believed that the binding of the antibodies of the present invention to the R3 domain may trigger a conformational or electrostatic change in the catalytic domain of glucosaminidase.

Inhibition of Gmd activity can be measured in vitro. According to one approach, Gmd is first pre-titered to determine the concentration that will yield about a 50% reduction in A₄₉₀ in 60 minutes. Then 50 μL of antibody diluted in PBST is added to each well of a 96-well microtiter plate followed by 50 μL of appropriately diluted Gmd, and the mixture allowed to incubate for 5 or more minutes, and finally 100 μL of 0.15% mL is added and the initial A₄₉₀ measured. The plate is incubated at 37° C. and the A₄₉₀ measured at 30 and 60 minutes. Percent inhibition is calculated as 100·(1−(Δ₆₀A₄₉₀ inhibitor/Δ₆₀A₄₉₀ no inhibitor control)).

In certain embodiments, the monoclonal antibody of the present invention possesses an ability to cause clustering or clumping of S. aureus, cell-independent lysis of S. aureus, or both. Examples of antibodies that possess an ability to cause clumping of S aureus include, without limitation, monoclonal antibodies 1C11, 1E12, 2D11, 3A8, and 3H6. One example of a lytic antibody is monoclonal antibody 1C11. This antibody binds to a unique epitope present in the R3 domain, displays between about 70 to about 80 percent Gmd inhibitory activity, and promotes cell-independent lysis of S. aureus.

The monoclonal antibodies of the present invention also inhibit in vivo growth of S. aureus. Inhibition of in vivo growth of S. aureus can be measured according to a number of suitable standards. In one such embodiment, the in vivo growth of S. aureus can be assessed according to a bioluminescence assay of the type described in the accompanying Examples. Specifically, bioluminescent S. aureus (Xen 29; ATCC 12600) (Francis et al., “Monitoring Bioluminescent Staphylococcus aureus Infections in Living Mice Using a Novel luxABCDE Construct,” Infect. Immun. 68(6):3594-600 (2000); see also Contag et al., “Photonic Detection of Bacterial Pathogens in Living Hosts,” Mol. Microbiol. 18(4):593-603 (1995), each of which is hereby incorporated by reference in its entirety) is used to dose a transtibial implant with 500,000 CFU prior to surgical implant. Five week old female BALB/cJ mice can receive an intraperitoneal injection of saline (n=10) or 1 mg of purified antibody in 0.25 ml saline 3 days prior to surgery. The mice can be imaged to assess bioluminescence on various days (e.g., 0, 3, 5, 7, 11, and 14) and a comparison of BLI images can be compared to assess whether the antibody inhibits in vivo growth of S. aureus relative to the saline control.

In one embodiment the monoclonal antibody of the present invention comprises a V_(H) domain comprising one of the following amino acid sequences:

EVQLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLE WIGVINPYNGDTTYSQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVY YCARNYDEYFDVWGTGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVT LGCXVKG (SEQ ID NO: 1); or EVQLQESGPVLVKPGASVKLSCKASGYTFTDYFMNWVKQSHGKSLE WIGVINPFNGGNRYNQNFKGKATLTVDKSSSTAYMELNSLTSEDSAV YYCARGDYDSPWFDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTN SMVTLGCLVKGYSXSQ (SEQ ID NO: 2), where X is any amino acid; or EVQLQESGGGFVKPGGSLKLSCAASGFTFSTYVMSWVRQTPEKRLEW VATISDGGGHTYYLDNVKGRFTISRDNAKNNLYLHMSHLKSEDTAMY YCARAYYGSSYDAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQT NSMVTLGCLVKG (SEQ ID NO: 3); or EVQLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLE WVAEIKDKTNNHATYYAESVKGRFTISRDVSKSRVFLQMNSLRPEDT GIYYCTSGPYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVT LGCLVKGYFPE (SEQ ID NO: 4); or QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVNQAPGKGLKW MGWINTYSGVPTYADDFKGRFVFSLETSASTAYLQINNLKNEDTATYF CAREEYSSGYAAWFPYWGQGTLVTVSA (SEQ ID NO: 5), where X is any amino acid; or XXQLXXSGXXXXXPGXXXKXSCXASGXTFXXXXMXWVXQXXXKX LXWXXXIXXXXXXXXXXYXXXXKGXXXXXXXXXXXXXXXXXXXL XXEDXXXYXCXXXXYXXXXXXXXXXWGXGTXXTVSXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXX, where X is any amino acid or deletion thereof; (consensus sequence SEQ ID NO: 6; see FIG. 13) or EVQLQESGXXXVXPGXSXKXSCXASGXTFXXXXMXWVXQXXXKXL EWXXXIXXXXXXXXXXYXXXXKGXXTXXXDXXXXXXXXXXXXLX XEDXXXYYCXXXXYXXXXXXXDXWGXGTXXTVSXAKTTPPSVYPL APGSAAQTNSMVTLGCXVKGXXXXX. where X is any amino acid or deletion thereof, (consensus sequence SEQ ID NO: 31; see FIG. 11) or XXQLXXSGXXLXXPGXXXKXSCXASGXTFXXXXMXWVXQXPXKGL XWXXXIXXXTXXXXXXYAXXXKGRFXXSXXXSXSXXXLQXNXLXX EDTXXYXCXXXXXXSGXXXXFXYWGQGTXXTVSXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXX, where X is any amino acid or deletion thereof; (consensus sequence SEQ ID NO: 7; see FIG. 15) or QXQLVQSGXEXKXPGXXVKXSCKASGYXFTTYGMXWVXQAPGXGL XWMGWXNTYXGXPTYAXXFXGRFVFSXXTSASTAYLQIXXLKXEDX AXYXCARXXXXXXXXXXXXYWGQGTLVTVSA. where X is any amino acid or deletion thereof. (consensus sequence SEQ ID NO: 35, see FIG. 17A)

In another embodiment the monoclonal antibody of the present invention comprises a V_(L) domain comprising one of the following amino acid sequences:

DIVMTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIY DTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLT FG (SEQ ID NO: 8); or QMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYY TSSLHSGVPSRFSGGGSGTDYSLSISNLEPEDIATYYCQQYSKLPWTFG GGTKLEIK, (SEQ ID NO: 9); or DIVITQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKXWI YSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPW TFGGGT (SEQ ID NO: 10); or DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLL IYWTSTRHTGVPDRFTGSGSGTDFTLTISSVQAKDLALYYCQQHYTTP YTFGGGTKLEIK, (SEQ ID NO: 11); or DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIEY ASRSISGIPSRFSGGGSGTDFTLSINSVESEDFGLYFCQQSNSWPLTFGA GTKLELK, (SEQ ID NO: 12); or DIXXTQXXXXXSXXXGXXVXXXCXASXXXSXXXXXWYQQKXXXX XXXXIXXXSXXXXGXPXRFXGXGSGTXXXLXIXXXXXXDXXXYXCX QXXXXPXTFGXGTXXXXX. where X is any amino acid or deletion thereof; (consensus sequence SEQ ID NO: 13, see FIG. 14A) or DIVXTQSXXXXSXXXGDXVSXXCXASQXXSXXXXWYQQKXXXSPXL LIXXXSXXXXGXPXRFXGXGSGTDFTLXIXSVXXXDXXLYXCQQXXX XPXTFGXGTKLEXK, where X is any amino acid or deletion thereof; (consensus sequence SEQ ID NO: 14, see FIG. 16) or DIVXTQSPAIMSASXGEXVTMTCXASSSVSXXYXXWYQQKPGSSPXX XIYXTSNLASGVPXRFSGSGSGTSYSLTISXMEAEDAATYYCXQXXXX PXTFGXXX, where Xis any amino acid or deletion thereof; (consensus sequence SEQ ID NO: 32, see FIG. 12) or DIXXTQXXXXXSXSXSXXSXXXSXSSXXXSXXXXXWYQQKPXXXXX XXIYXTSXXXXCGVPPXRFXGXGSGTXXXLXISXXXXXDXAXYYCXQ XXXXPXTFGGGTXXXXX, where X is any amino acid or deletion thereof; (consensus sequence SEQ ID NO: 33, see FIG. 14B) or XIVLTQSPXXXSVTPXXXVXXXCRASQSIXXXLHWYQQXXXXSPXLLI XYASXSXSGXPSRFSGXGSGTDFTLXINSXEXEDXXXYXCXQSXSXPL TFGXGTKXEXK, where X is any amino acid or deletion thereof. (consensus sequence SEQ ID NO: 37, see FIG. 17B)

Antibodies of the present invention may also be synthetic antibodies. A synthetic antibody is an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. Alternatively, the synthetic antibody is generated by the synthesis of a DNA molecule encoding and expressing the antibody of the invention or the synthesis of an amino acid specifying the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

The monoclonal antibody of the present invention can be humanized. Humanized antibodies are antibodies that contain minimal sequences from non-human (e.g. murine) antibodies within the variable regions. Such antibodies are used therapeutically to reduce antigenicity and human anti-mouse antibody responses when administered to a human subject. In practice, humanized antibodies are typically human antibodies with minimum to no non-human sequences. A human antibody is an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.

An antibody can be humanized by substituting the complementarity determining region (CDR) of a human antibody with that of a non-human antibody (e.g. mouse, rat, rabbit, hamster, etc.) having the desired specificity, affinity, and capability (Jones et al., “Replacing the Complementarity-Determining Regions in a Human Antibody With Those From a Mouse,” Nature 321:522-525 (1986); Riechmann et al., “Reshaping Human Antibodies for Therapy,” Nature 332:323-327 (1988); Verhoeyen et al., “Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science 239:1534-1536 (1988), which are hereby incorporated by reference in their entirety). The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability.

Humanized antibodies can be produced using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated (see e.g. Reisfeld et al., MONOCLONAL ANTIBODIES AND CANCER THERAPy 77 (Alan R. Liss ed., 1985) and U.S. Pat. No. 5,750,373 to Garrard, which are hereby incorporated by reference in their entirety). Also, the humanized antibody can be selected from a phage library, where that phage library expresses human antibodies (Vaughan et al., “Human Antibodies with Sub-Nanomolar Affinities Isolated from a Large Non-immunized Phage Display Library,” Nature Biotechnology, 14:309-314 (1996); Sheets et al., “Efficient Construction of a Large Nonimmune Phage Antibody Library: The Production of High-Affinity Human Single-Chain Antibodies to Protein Antigens,” Proc. Nat'l. Acad. Sci. U.S.A. 95:6157-6162 (1998); Hoogenboom et al., “By-passing Immunisation. Human Antibodies From Synthetic Repertoires of Germline VH Gene Segments Rearranged in vitro,” J. Mol. Biol. 227:381-8 (1992); Marks et al., “By-passing Immunization. Human Antibodies from V-gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-97 (1991), which are hereby incorporated by reference in their entirety). Humanized antibodies can also be made in transgenic mice containing human immunoglobulin loci that are capable upon immunization of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Pat. No. 5,545,807 to Surani et al.; U.S. Pat. No. 5,545,806 to Lonberg et al.; U.S. Pat. No. 5,569,825 to Lonberg et al.; U.S. Pat. No. 5,625,126 to Lonberg et al.; U.S. Pat. No. 5,633,425 to Lonberg et al.; and U.S. Pat. No. 5,661,016 to Lonberg et al., which are hereby incorporated by reference in their entirety.

Based on a BLAST search of Genbank using the 1C11 V. and V_(L) domain amino acid sequences, homologous sequences within the human genome were identified as IGVH7-81 and IGVK6D-21, respectively. Alignments of these homologous V. and V_(L) domains (SEQ ID NOS: 19 and 20, respectively) with the corresponding 1C11 V. and V_(L) domains are illustrated in FIGS. 17A-B, respectively. The V_(H) and V_(L) domains share a surprisingly high degree of identity, respectively about 75% and 67% over the region of homology (i.e., excluding V. CDR3H region). These IGVH7-81 and IGVK6D-21 sequences can be used to prepare a substantially pure monoclonal antibody of the present invention. Because the CDR3H is not encoded by the V. gene, a suitable D region will need to be spliced into the missing domain region. Any one of several candidate D regions can be used (e.g., IGHD5-5, 18 or 12*01).

In addition to whole antibodies, the present invention encompasses binding portions of such antibodies. Such binding portions include the monovalent Fab fragments, Fv fragments (e.g., single-chain antibody, scFv), and single variable V_(H) and V_(L) domains, and the bivalent F(ab′)₂ fragments, Bis-scFv, diabodies, triabodies, minibodies, etc. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in James Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (Academic Press, 1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Laboratory, 1988); Houston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Bird et al, “Single-Chain Antigen-Binding Proteins,” Science 242:423-426 (1988), which are hereby incorporated by reference in their entirety, or other methods known in the art.

It may further be desirable, especially in the case of antibody fragments, to modify the antibody to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope binding site into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis).

Antibody mimics are also suitable for use in accordance with the present invention. A number of antibody mimics are known in the art including, without limitation, those known as monobodies, which are derived from the tenth human fibronectin type III domain (¹⁰Fn3) (Koide et al., “The Fibronectin Type III Domain as a Scaffold for Novel Binding Proteins,” J. Mol. Biol. 284:1141-1151 (1998); Koide et al., “Probing Protein Conformational Changes in Living Cells by Using Designer Binding Proteins: Application to the Estrogen Receptor,” Proc. Natl. Acad. Sci. USA 99:1253-1258 (2002), each of which is hereby incorporated by reference in its entirety); and those known as affibodies, which are derived from the stable alpha-helical bacterial receptor domain Z of staphylococcal protein A (Nord et al., “Binding Proteins Selected from Combinatorial Libraries of an alpha-helical Bacterial Receptor Domain,” Nature Biotechnol. 15(8):772-777 (1997), which is hereby incorporated by reference in its entirety).

In preparing these antibody mimics the CDR sequences of the V_(H) and/or V_(L) chains can be grafted into the variable loop regions of these antibody mimics (see FIGS. 11 and 17 for putative CDR domains). The grafting can involve a deletion of at least two amino acid residues up to substantially all but one amino acid residue appearing in a particular loop region along with the substitution of the CDR sequence. Insertions can be, for example, an insertion of the CDR domain at one or more locations of a particular loop region. The antibody mimics of the present invention preferably possess an amino acid sequence which is at least 50% homologous to the V_(H) and/or V_(L) chains sequences disclosed in the present application. The deletions, insertions, and replacements on the polypeptides can be achieved using recombinant techniques beginning with a known nucleotide sequence (see infra).

Methods for monoclonal antibody production may be achieved using the techniques described herein or other well-known in the art (MONOCLONAL ANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporated by reference in its entirety). Generally, the process involves obtaining immune cells (lymphocytes) from the spleen of a mammal which has been previously immunized with the antigen of interest (i.e., S. aureus glucosaminidase or peptide fragments thereof).

The antibody-secreting lymphocytes are then fused with myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is achieved by standard and well-known techniques, for example, by using polyethylene glycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation of Specific Antibody-Producing Tissue Culture and Tumor Lines by Cell Fusion,” Eur. J. Immunol. 6:511 (1976), which is hereby incorporated by reference in its entirety). The immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and have good fusion capability. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody.

Thus, a second aspect of present invention relates to a cell line that expresses a monoclonal antibody of the present invention. In one embodiment the monoclonal antibody of the present invention is produced by a hybridoma cell line designated as 1C11, 1E12, 2D11, 3A8, or 3H6. In another embodiment, the monoclonal antibody of the present invention (or a binding portion thereof) is produced by a recombinant cell or cell line.

As noted above, monoclonal antibodies can be made using recombinant DNA methods as described in U.S. Pat. No. 4,816,567 to Cabilly et al., which is hereby incorporated by reference in its entirety. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, generate host cells that express and secrete monoclonal antibodies. Also, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries (McCafferty et al., “Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains,” Nature 348:552-554 (1990); Clackson et al., “Making Antibody Fragments using Phage Display Libraries,” Nature 352:624-628 (1991); and Marks et al., “By-Passing Immunization. Human Antibodies from V-Gene Libraries Displayed on Phage,” J. Mol. Biol. 222:581-597 (1991), which are hereby incorporated by reference in their entirety).

The present invention also includes a nucleic acid molecule encoding a polypeptide of the present invention. In one embodiment the nucleic acid is DNA. Examples of such DNA sequences are those that comprise a V_(H) and/or V_(L) encoding sequence of the present invention. DNA sequence encoding for hybridoma 2D11 V_(H) (closest germ line match: J558.18.108) has the nucleotide sequence (SEQ ID NO: 21) as follows:

GAGGTGCAGCTGCAGGAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTC AGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTATA TGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTT ATTAATCCTTACAACGGTGATACTACCTACAGCCAGAAGTTCAAGGGCAA GGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCA ACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAATTAC GACGAGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTC AGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTG CCCAAACTAACTCCATGGTGACCCTGGGATGCCNGGTCAAGGGC

DNA sequence encoding for hybridoma 3H6 V_(H) (closest germ line match: J558.18.108) has the nucleotide sequence (SEQ ID NO: 23) as follows:

GAGGTGCAGCTGCAGGAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTC AGTGAAGCTGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTTTA TGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTT ATTAATCCTTTCAACGGTGGTAATAGGTACAACCAGAACTTCAAGGGCAA GGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCA ACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGGAC TATGACTCCCCCTGGTTTGATTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGAT CTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGC TATTCCCNGAGCCAGTG

DNA sequence encoding for hybridoma 1E12 V_(H) (closest germ line match: 7183.46 VH7) has the nucleotide sequence (SEQ ID NO: 25) as follows:

GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTCGTGAAGCCTGGAGGGTC CCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGTCA TGTCTTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACC ATTAGTGATGGTGGTGGTCATACTTACTATCTAGACAATGTAAAGGGCCG ATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCACATGA GCCATCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGCTTAC TACGGTAGTAGTTACGACGCTATGGACTACTGGGGTCAAGGAACCTCAGT CACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCC CTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTC AAGGGC

DNA sequence encoding for hybridoma 3A8 V_(H) (closest germ line match: VHJ606.4.8.2) has the nucleotide sequence (SEQ ID NO: 27) as follows:

GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATC CATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGA TGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAA ATTAAAGACAAAACTAATAATCATGCAACATACTATGCTGAGTCTGTGAA AGGGAGGTTCACCATCTCAAGAGATGTTTCCAAAAGTCGTGTCTTCCTGC AAATGAACAGCTTAAGACCTGAAGACACTGGCATTTATTACTGTACGTCT GGGCCATATTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTC AGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTG CCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTC CCTGAG

DNA sequence encoding for hybridoma 1C11 V_(H) (closest germline match: VH 9-15, DST4-057B1-6, JH3) has the nucleotide sequence (SEQ ID NO: 29) as follows:

CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGAC AGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAACGTATGGAA TGAGCTGGGTGAATCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGG ATAAACACCTACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGACG GTTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCA ACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGAGGAG TACAGCTCAGGCTACGCGGCCTGGTTTCCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCTGCA

DNA sequence encoding for the 2D11 V_(L) (closest germ line match: at4) has the nucleotide sequence (SEQ ID NO: 22) as follows:

GATATTGTGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGA GAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACT GGTACCAGCAGAAGCCAGGATCCTCCCCCAGACTCCTGATTTATGACACA TCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGG GACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAGGATGCTGCCA CTTATTACTGCCAGCAGTGGAGTAGTTACCCGCTCACGTTCGGT

DNA sequence encoding for the 3H6 V_(L) (closest germ line match: cp9, JK1) has the nucleotide sequence (SEQ ID NO: 24) as follows:

CAGATGACACAGACTACGTCCTCCCTGTCTGCCTCTCTGGGAGACAGAGT CACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGT ATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCA AGTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCGGTGGGTCTGGGAC AGATTATTCTCTCTCCATCAGCAACCTGGAACCTGAAGATATTGCCACTT ACTATTGTCAGCAGTATAGTAAGCTTCCTTGGACGTTCGGTGGAGGCACC AAGCTGGAAATCAAA

DNA sequence encoding for the 1E12 V_(L) (closest germ line match: ai4) has the nucleotide sequence (SEQ ID NO: 26) as follows:

GATATTGTGATCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGA ACGGGTCACCATGACCTGCACTGCCAGCTCAAGTGTAAGTTCCAGTTACT TACACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTNTGGATTTAT AGCACATCCAACCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGG GTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATG CTGCCACTTATTACTGCCACCAGTATCATCGTTCCCCATGGACGTTCGGT GGAGGCACC

DNA sequence encoding for the 3A8 V_(L) (closest germ line match: KV 19-25, JK2) has the nucleotide sequence (SEQ ID NO: 28) as follows:

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGA CAGGGTCAGCATCACCTGCAAGGCCAGTCAGGACGTGAGTACTGCTGTAG CCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGG ACATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATC TGGGACAGATTTTACTCTCACCATCAGCAGTGTGCAGGCTAAAGACCTGG CACTTTATTACTGTCAGCAACATTATACCACTCCGTACACGTTCGGAGGG GGGACCAAGCTGGAAATAAAA

DNA sequence encoding for 1C11 V_(L) (closest germ line match: VK23-43, JK5) has the nucleotide sequence (SEQ ID NO: 30) as follows:

GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACAACCTAC ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCGAATAT GCTTCCCGGTCCATCTCTGGGATCCCCTCTAGGTTCAGTGGCGGTGGATC AGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGTCTGAAGATTTTG GATTGTATTTCTGTCAACAGAGTAACAGCTGGCCGCTCACGTTCGGTGCT GGGACCAAGCTGGAGCTGAAA

Still a further aspect of the present invention is a DNA construct comprising a DNA molecule that encodes an antibody or binding portion of the present invention, a promoter-effective DNA molecule operably coupled 5′ of the DNA molecule, and a transcription termination DNA molecule operably coupled 3′ of the DNA molecule. The present invention also encompasses an expression vector into which the DNA construct of the present invention is inserted. A synthetic gene for the polypeptides of the present invention can be designed such that it includes convenient restriction sites for ease of mutagenesis and uses specific codons for high-level protein expression (Gribskov et al., “The Codon Preference Plot: Graphic Analysis of Protein Coding Sequences and Prediction of Gene Expression,” Nuc. Acids. Res. 12:539-549 (1984), which is hereby incorporated by reference in its entirety).

The gene may be assembled as follows: first the gene sequence can be divided into parts with boundaries at designed restriction sites; for each part, a pair of oligonucleotides that code opposite strands and have complementary overlaps of about 15 bases can be synthesized; the two oligonucleotides can be annealed and single strand regions can be filled in using the Klenow fragment of DNA polymerase; the double-stranded oligonucleotide can be cloned into a vector, such as, the pET3a vector (Novagen) using restriction enzyme sites at the termini of the fragment and its sequence can be confirmed by a DNA sequencer; and these steps can be repeated for each of the parts to obtain the whole gene. This approach takes more time to assemble a gene than the one-step polymerase chain reaction (PCR) method (Sandhu et al., “Dual Asymetric PCR: One-Step Construction of Synthetic Genes,” BioTech. 12:14-16 (1992), which is hereby incorporated by reference in its entirety). Mutations could likely be introduced by the low fidelity replication by Taq polymerase and would require time-consuming gene-editing. Recombinant DNA manipulations can be performed according to SAMBROOK & RUSSELL, M OLECULAR CLONING: A LABORATORY MANUAL (2d ed. 1989), which is hereby incorporated by reference in its entirety, unless otherwise stated. To avoid the introduction of mutations during one-step PCR, high fidelity/low error polymerases can be employed as is known in the art.

Desired mutations can be introduced to the polypeptides sequence of the present invention using either cassette mutagenesis, oligonucleotide site-directed mutagenesis techniques (Deng & Nickoloff, “Site-Directed Mutagenesis of Virtually any Plasmid by Eliminating a Unique Site,” Anal. Biochem. 200:81-88 (1992), which is hereby incorporated by reference in its entirety), or Kunkel mutagenesis (Kunkel et al., “Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection,” Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., “Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection,” Methods Enzymol. 154:367-382 (1987), which are hereby incorporated by reference in their entirety).

Both cassette mutagenesis and site-directed mutagenesis can be used to prepare specifically desired nucleotide coding sequences. Cassette mutagenesis can be performed using the same protocol for gene construction described above and the double-stranded DNA fragment coding a new sequence can be cloned into a suitable expression vector. Many mutations can be made by combining a newly synthesized strand (coding mutations) and an oligonucleotide used for the gene synthesis. Regardless of the approach utilized to introduce mutations into the nucleotide sequence encoding a polypeptide according to the present invention, sequencing can be performed to confirm that the designed mutations (and no other mutations) were introduced by mutagenesis reactions.

In contrast, Kunkel mutagenesis can be utilized to randomly produce a plurality of mutated polypeptide coding sequences which can be used to prepare a combinatorial library of polypeptides for screening. Basically, targeted loop regions (or C-terminal or N-terminal tail regions) can be randomized using the NNK codon (N denoting a mixture of A, T, G, C, and K denoting a mixture of G and T) (Kunkel et al., “Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection,” Methods Enzymol. 154:367-382 (1987), which is hereby incorporated by reference in its entirety).

Regardless of the approach used to prepare the nucleic acid molecules encoding the polypeptide according to the present invention, the nucleic acid can be incorporated into host cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e., not normally present). The heterologous DNA molecule is inserted into the expression system or vector in sense orientation and correct reading frame. The vector contains the necessary elements (promoters, suppressers, operators, transcription termination sequences, etc.) for the transcription and translation of the inserted protein-coding sequences. A recombinant gene or DNA construct can be prepared prior to its insertion into an expression vector. For example, using conventional recombinant DNA techniques, a promoter-effective DNA molecule can be operably coupled 5′ of a DNA molecule encoding the polypeptide and a transcription termination (i.e., polyadenylation sequence) can be operably coupled 3′ thereof.

In accordance with this aspect of the invention, the polynucleotides of the present invention are inserted into an expression system or vector to which the molecule is heterologous. The heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5′→3′) orientation relative to the promoter and any other 5′ regulatory molecules, and correct reading frame. The preparation of the nucleic acid constructs can be carried out using standard cloning methods well known in the art as described by SAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press, 2001), which is hereby incorporated by reference in its entirety. U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference in its entirety, also describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase.

Suitable expression vectors include those which contain replicon and control sequences that are derived from species compatible with the host cell. For example, if E. coli is used as a host cell, plasmids such as pUC19, pUC18 or pBR322 may be used. When using insect host cells, appropriate transfer vectors compatible with insect host cells include, pVL1392, pVL1393, pAcGP67 and pAcSecG2T, which incorporate a secretory signal fused to the desired protein, and pAcGHLT and pAcHLT, which contain GST and 6×His tags (BD Biosciences, Franklin Lakes, N.J.). Viral vectors suitable for use in carrying out this aspect of the invention include, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, nodaviral vectors, and retroviral vectors. Other suitable expression vectors are described in SAMBROOK AND RUSSELL, M OLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press, 2001), which is hereby incorporated by reference in its entirety. Many known techniques and protocols for manipulation of nucleic acids, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Fred M. Ausubel et al. eds., 2003), which is hereby incorporated by reference in its entirety.

Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation) and subsequently the amount of antibodies or antibody fragments that are produced and expressed by the host cell. Transcription of DNA is dependent upon the presence of a promoter, which is a DNA sequence that directs the binding of RNA polymerase, and thereby promotes mRNA synthesis. Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters to obtain a high level of transcription and, hence, expression. Depending upon the host system utilized, any one of a number of suitable promoters may be used. For instance, when using E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L) promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV 5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene. When using insect cells, suitable baculovirus promoters include late promoters, such as 39K protein promoter or basic protein promoter, and very late promoters, such as the p10 and polyhedron promoters. In some cases it may be desirable to use transfer vectors containing multiple baculoviral promoters. Common promoters suitable for directing expression in mammalian cells include, without limitation, SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR. The promoters can be constitutive or, alternatively, tissue-specific or inducible. In addition, in some circumstances inducible (TetOn) promoters can be used.

Translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals, which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, “Maximizing Gene Expression on a Plasmid Using Recombination In Vitro,” Methods in Enzymology, 68:473-82 (1979), which is hereby incorporated by reference in its entirety.

The present invention also includes a host cell transformed with the DNA construct of the present invention. The host cell can be a prokaryote or a eukaryote. Host cells suitable for expressing the polypeptides of the present invention include any one of the more commonly available gram negative bacteria. Suitable microorganisms include Pseudomonas aeruginosa, Escherichia coli, Salmonella gastroenteritis (typhimirium), S. typhi, S. enteriditis, Shigella flexneri, S. sonnie, S. dysenteriae, Neisseria gonorrhoeae, N meningitides, Haemophilus influenzae, H pleuropneumoniae, Pasteurella haemolytica, P. multilocida, Legionella pneumophila, Treponema pallidum, T. denticola, T. orales, Borrelia burgdorferi, Borrelia spp., Leptospira interrogans, Klebsiella pneumoniae, Proteus vulgaris, P. morganii, P. mirabilis, Rickettsia prowazeki, R. typhi, R. richettsii, Porphyromonas (Bacteroides) gingivalis, Chlamydia psittaci, C. pneumoniae, C. trachomatis, Campylobacter jejuni, C. intermedis, C. fetus, Helicobacter pylori, Francisella tularenisis, Vibrio cholerae, Vibrio parahaemolyticus, Bordetella pertussis, Burkholderie pseudomallei, Brucella abortus, B. susi, B. melitensis, B. canis, Spirillum minus, Pseudomonas mallei, Aeromonas hydrophila, A. salmonicida, and Yersinia pestis.

In addition to bacteria cells, animal cells, in particular mammalian and insect cells, yeast cells, fungal cells, plant cells, or algal cells are also suitable host cells for transfection/transformation of the recombinant expression vector carrying an isolated polynucleotide molecule of the present invention. Mammalian cell lines commonly used in the art include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells, and many others. Suitable insect cell lines include those susceptible to baculoviral infection, including Sf9 and Sf21 cells.

Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected, as described in SAMBROOK & RUSSELL, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Springs Laboratory Press, 2001), which is hereby incorporated by reference in its entirety. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. For eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or any other viral vector. For insect cells, the transfer vector containing the polynucleotide construct of the present invention is co-transfected with baculovirus DNA, such as AcNPV, to facilitate the production of a recombinant virus. Subsequent recombinant viral infection of Sf cells results in a high rate of recombinant protein production. Regardless of the expression system and host cell used to facilitate protein production, the expressed antibodies, antibody fragments, or antibody mimics of the present invention can be readily purified using standard purification methods known in the art and described in PHILIP L. R. BONNER, PROTEIN PURIFICATION (Routledge 2007), which is hereby incorporated by reference in its entirety.

The polynucleotide(s) encoding a monoclonal antibody can further be modified using recombinant DNA technology to generate alternative antibodies. For example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be substituted for those regions of a human antibody to generate a humanized (or chimeric) antibody, as discussed above. Alternatively, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be substituted for a non-immunoglobulin polypeptide to generate a fusion antibody. In other embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Furthermore, site-directed or high-density mutagenesis of the variable region can be used to optimize specificity and affinity of a monoclonal antibody.

A third aspect of the present invention is related to a pharmaceutical composition comprising a carrier and one or more monoclonal antibodies or one or more binding portions thereof in accordance with the present invention. This pharmaceutical composition may contain two or more antibodies or binding fragments where all antibodies or binding fragments recognize the same epitope. Alternatively, the pharmaceutical composition may contain an antibody or binding fragment mixture where one or more antibodies or binding fragments recognize one epitope of S. aureus Gmd and one or more antibodies or binding fragments recognize a different epitope of S. aureus Gmd. For example, the mixture may contain one or more antibodies of the present invention that bind specifically to an R3 domain of Staphylococcus aureus glucosaminidase in combination with any other antibody that binds to glucosaminidase, such as an antibody that binds to the catalytic domain of glucosaminidase. The pharmaceutical composition of the present invention further contains a pharmaceutically acceptable carrier or other pharmaceutically acceptable components as described infra

In accordance with one embodiment, the pharmaceutical composition includes one or more of mAbs 1C11, 2D11, 3H6, 1E12, and 3A8 in a pharmaceutically acceptable carrier. In accordance with another embodiment, the pharmaceutical composition includes two or more of mAbs 1C11, 2D11, 3H6, 1E12, and 3A8 in a pharmaceutically acceptable carrier.

A pharmaceutical composition containing the monoclonal antibodies of the present invention can be administered to a subject having or at risk of having Staphylococcus infection. Various delivery systems are known and can be used to administer the antibodies of the present invention. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The therapeutic agent can be administered, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, and the like) and can be administered together with other biologically active agents, such as chemotherapeutic agents, antibiotic agents, or other immunotherapeutic agents. Administration can be systemic or local, i.e., at a site of Staph infection or directly to a surgical or implant site.

The pharmaceutical composition of the present invention can further comprise administering a second therapeutic agent to the patient, wherein the second therapeutic agent is an antibiotic agent or immunotherapeutic agent. Exemplary antibiotic agents include, without limitation, vancomycin, tobramycin, cefazolin, erythromycin, clindamycin, rifampin, gentamycin, fusidic acid, minocycline, co-trimoxazole, clindamycin, linezolid, quinupristin-dalfopristin, daptomycin, tigecycline, dalbavancin, telavancin, oritavancin, ceftobiprole, ceftaroline, iclaprim, the carbapenem CS-023/RO-4908463, and combinations thereof. Exemplary immunotherapeutic agents include, without limitation, tefibazumab, BSYX-A110, Aurexis™, and combinations thereof. The above lists of antibiotic agents and immunotherapeutic agents are intended to be non-limiting examples; thus, other antibiotic agents or immunotherapeutic agents are also contemplated. Combinations or mixtures of the second therapeutic agent can also be used for the purposes of the present invention. These agents can be administered contemporaneously or as a single formulation.

The pharmaceutical composition typically includes one or more pharmaceutical carriers (e.g., sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the nucleic acid or protein, typically in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulations correspond to the mode of administration.

Effective doses of the compositions of the present invention, for the treatment of the above described bacterial infections vary depending upon many different factors, including mode of administration, target site, physiological state of the patient, other medications administered, and whether treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime. For prophylactic treatment against Staphylococcus bacterial infection, it is intended that the pharmaceutical composition(s) of the present invention can be administered prior to exposure of an individual to the bacteria and that the resulting immune response can inhibit or reduce the severity of the bacterial infection such that the bacteria can be eliminated from the individual. For example, the monoclonal antibody or the pharmaceutical composition can be administered prior to, during, and/or immediately following a surgical procedure, such as joint replacement or any surgery involving a prosthetic implant.

For passive immunization with an antibody or binding fragment of the present invention, the dosage ranges from about 0.0001 to about 100 mg/kg, and more usually about 0.01 to about 5 mg/kg, of the host body weight. For example, dosages can be about 1 mg/kg body weight or about 10 mg/kg body weight, or within the range of about 1 to about 10 mg/kg. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Antibody is usually administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly, or yearly. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies.

A further aspect of the invention relates to an active vaccine (e.g., pharmaceutical composition) that includes a carrier and an antigenic molecule comprising at least a fragment of the S. aureus glucosaminidase. The antigenic molecule can be in the form of (i) a fusion protein that includes the glucosaminidase polypeptide and an adjuvant polypeptide or (ii) an immunogenic conjugate that includes the glucosaminidase polypeptide conjugated to another immunogenic molecule.

By way of example, and without limitation, suitable fusion proteins of the present invention include those containing an adjuvant polypeptide selected from the group of flagellin, human papillomavirus (HPV) L1 or L2 proteins, herpes simplex glycoprotein D (gD), complement C4 binding protein, a toll-like receptor-4 (TLR4) ligand, and IL-1β.

The fusion polypeptide or protein of the present invention can be generated using standard techniques known in the art. For example, the fusion polypeptide can be prepared by translation of an in-frame fusion of the polynucleotide sequences of the present invention and the adjuvant, i.e., a hybrid or chimeric gene. The hybrid gene encoding the fusion polypeptide is inserted into an expression vector which is used to transform or transfect a host cell. Alternatively, the polynucleotide sequence encoding the polypeptide of the present invention is inserted into an expression vector in which the polynucleotide encoding the adjuvant is already present. The peptide adjuvant of the fusion protein can be fused to the N-, or preferably, to the C-terminal end of the glucosaminidase polypeptide of the present invention.

Fusions between the polypeptides of the present invention and the protein adjuvant may be such that the amino acid sequence of the polypeptide of the present invention is directly contiguous with the amino acid sequence of the adjuvant. Alternatively, the polypeptide portion may be coupled to the adjuvant by way of a short linker sequence. Suitable linker sequences include glycine rich linkers (e.g., GGGS₂₋₃ (SEQ ID NO: 37), serine-rich linkers (e.g., GS_(N)), or other flexible immunoglobulin linkers as disclosed in U.S. Pat. No. 5,516,637 to Huang et al, which is hereby incorporated by reference in its entirety.

Suitable immunogenic conjugates of the present invention include, but are not limited to, those containing an immunogenic carrier molecule covalently or non-covalently bonded to any glucosaminidase polypeptide. Any suitable immunogenic carrier molecule can be used. Exemplary immunogenic carrier molecules include, but are in no way limited to, bovine serum albumin, chicken egg ovalbumin, keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid, thyroglobulin, a pneumococcal capsular polysaccharide, CRM 197, and a meningococcal outer membrane protein.

The pharmaceutical composition in the form of an active vaccine can also include an effective amount of a separate adjuvant. Suitable adjuvants for use in the present invention include, without limitation, aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid, Quil A, and/or non-infective Bordetella pertussis.

The choice of an adjuvant depends on the stability of the immunogenic formulation containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the species being vaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, alum, MPL or Incomplete Freund's adjuvant (Chang et al., Advanced Drug Delivery Reviews 32:173-186 (1998), which is hereby incorporated by reference in its entirety) alone or optionally all combinations thereof are suitable for human administration.

In prophylactic applications, pharmaceutical compositions containing the immunogenic glucosaminidase polypeptides are administered to a patient susceptible to, or otherwise at risk of, the bacterial infection in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presented during development of the disease. In therapeutic applications, pharmaceutical compositions containing a monoclonal antibody or binding fragment according to the present invention are administered to a patient suspected of, or already suffering from, such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose, which is identified supra. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient response has been achieved. Typically, the response is monitored and repeated dosages are given if the response starts to wane.

Treatment dosages should be titrated to optimize safety and efficacy. The amount of immunogen depends on whether adjuvant is also administered, with higher dosages being required in the absence of adjuvant. The amount of an immunogen for administration sometimes varies from 1-500 μg per patient and more usually from 5-500 μg per injection for human administration. Occasionally, a higher dose of 1-2 mg per injection is used. Typically about 10, 20, 50, or 100 μg is used for each human injection. The mass of immunogen also depends on the mass ratio of immunogenic epitope within the immunogen to the mass of immunogen as a whole. Typically, 10⁻³ to 10⁻⁵ micromoles of immunogenic epitope are used for each microgram of immunogen. The timing of injections can vary significantly from once a day, to once a year, to once a decade. On any given day that a dosage of immunogen is given, the dosage is greater than 1 μg/patient and usually greater than 10 μg/patient if adjuvant is also administered, and greater than 10 μg/patient and usually greater than 100 μg/patient in the absence of adjuvant. A typical regimen consists of an immunization followed by booster injections at time intervals, such as 6 week intervals. Another regimen consists of an immunization followed by booster injections 1, 2, and 12 months later. Alternatively, booster injections can be provided on a regular or irregular basis, as indicated by monitoring of immune response.

A fourth aspect the present invention relates to a method of treating an S. aureus infection that includes administering to a patient having an S. aureus infection an effective amount of a monoclonal antibody or binding fragment thereof or a pharmaceutical composition of the present invention.

In one embodiment of this aspect of the invention the method of treating S. aureus infection further comprises repeating said administering. The method of treating S. aureus infection can be such that the administering is carried out systemically or carried out directly to a site of the S. aureus infection.

The method of treating S. aureus infection can be used to treat S. aureus infection at sites which include, without limitation, infection of the skin, muscle, cardiac, respiratory tract, gastrointestinal tract, eye, kidney and urinary tract, and bone or joint infections.

In one embodiment, this method is carried out to treat osteomyelitis by administering an effective amount of the monoclonal antibody or binding fragment thereof or the pharmaceutical composition of the present invention to a patient having an S. aureus bone or joint infection. Administration of these agents or compositions can be carried out using any of the routes described supra; however, administration directly to the site of the bone or joint infection is preferred.

A sixth aspect of the present invention relates to a method of introducing an orthopedic implant into a patient that includes administering to a patient in need of an orthopedic implant an effective amount of a monoclonal antibody, binding portion, or pharmaceutical composition of the present invention, and introducing the orthopedic implant into the patient.

In one embodiment, the method of introducing an orthopedic implant includes administering to the patient in need of the orthopedic implant an effective amount of a monoclonal antibody or binding fragment or a pharmaceutical composition containing the same, directly to the site of implantation. Alternatively, or in addition, the orthopedic implant can be coated or treated with the monoclonal antibody or binding fragment or a pharmaceutical composition containing the same before, during, or immediately after implantation thereof at the implant site.

The orthopedic implant can be a joint prosthesis, graft or synthetic implant. Exemplary joint prosthetics includes, without limitation, a knee prosthetic, hip prosthetic, finger prosthetic, elbow prosthetic, shoulder prosthetic, temperomandibular prosthetic, and ankle prosthetic. Other prosthetics can also be used. Exemplary grafts or synthetic implants include, without limitation, an artificial intervertebral disk, meniscal implant, or a synthetic or allograft anterior cruciate ligament, medial collateral ligament, lateral collateral ligament, posterior cruciate ligament, Achilles tendon, and rotator cuff. Other grafts or implants can also be used.

In one embodiment, the method of introducing an orthopedic implant is intended to encompass the process of installing a revision total joint replacement. Where infection, particularly Staph infection of an original joint replacement occurs, the only viable treatment is a revision total joint replacement. In this embodiment, the infected joint prosthesis is first removed and then the patient is treated for the underlying infection. Treatment of the infection occurs over an extended period of time (i.e. 6 months), during which time the patient is immobile (or has only limited mobility) and receives high doses of antibiotics to treat the underlying infection and optionally one or more monoclonal antibodies or binding portions, or pharmaceutical compositions of the present invention. Upon treatment of the underlying infection, the new joint prosthesis is installed. Immediately prior (i.e., within the two weeks preceding new joint prosthesis installation) and optionally subsequent to installation of the new joint prosthesis, the patient is administered one or more monoclonal antibodies or binding portions, or pharmaceutical compositions of the present invention. This treatment can be repeated one or more times during the post-installation period. Antibiotic treatment may be administered in combination with or concurrently with the one or more monoclonal antibodies or binding portions, or pharmaceutical compositions of the present invention. These treatments are effective to prevent infection or reinfection during the revision total joint replacement.

The methods of treatment according to the present invention can be used to treat any patient in need, however, the methods are particularly useful for immuno-compromised patients of any age, as well as patients that are older than 50 years of age.

EXAMPLES

The present invention is illustrated by reference to the following examples. These examples are not intended to limit the claimed invention.

Example 1—A Murine Transtibial Model of Implant-Associated Osteomyelitis

Orthopedic implant-associated OM occurs for both intramedullary devices (i.e. joint prostheses) and transcortical implants (i.e. external fixation devices, FIG. 1A). Although the infection rate of fixation devices is 2.5 times greater, and has an incidence of over 8-times that of total joint prostheses, it is not considered to be as serious because the revision surgery is much simpler (Darouiche, “Treatment of Infections Associated With Surgical Implants,” N Engl. J. Med. 350(14):1422-9 (2004), which is hereby incorporated by reference in its entirety). While most cases involving an infected transcortical implant can be resolved in a single surgery to relocate the pin and treating the abscess independently, the majority of infected prostheses must undergo two revision surgeries (Darouiche, “Treatment of Infections Associated With Surgical Implants,” N Engl. J. Med. 350(14):1422-9 (2004), which is hereby incorporated by reference in its entirety). The first is needed to cure the infection, and the second replaces the prosthesis. Thus, from a clinical significance standpoint, the field has focused primarily on models of implant-associated OM that involve an intramedullary device with the UAMS-1 (ATCC 49230) strain of S. aureus (Daum et al., “A Model of Staphylococcus aureus Bacteremia, Septic Arthritis, and Osteomyelitis in Chickens,” J. Orthop. Res. 8(6):804-13 (1990); Rissing et al., “Model of Experimental Chronic Osteomyelitis in Rats,” Infect. Immun. 47(3):581-6 (1985); Passl et al., “A Model of Experimental Post-Traumatic Osteomyelitis in Guinea Pigs,” J. Trauma 24(4):323-6 (1984); Worlock et al., “An Experimental Model of Post-Traumatic Osteomyelitis in Rabbits,” Br. J. Exp. Pathol. 69(2):235-44 (1988); Varshney et al., “Experimental Model of Staphylococcal Osteomyelitis in Dogs,” Indian J. Exp. Biol. 27(9):816-9 (1989); Kaarsemaker et al., “New Model for Chronic Osteomyelitis With Staphylococcus aureus in Sheep,” Clin. Orthop. Relat. Res. 339:246-52 (1997), which are hereby incorporated by reference in their entirety). Unfortunately, this approach has significant limitations, most notably the inability to generate reproducible (temporal and spatial) lesions. In an effort to overcome this the location of the infection was guided to the diaphysis by fracturing the tibia immediately after inserting an intramedullary pin containing 1×10⁶ CFU, using an Einhorn device as described previously (Zhang et al., “Cyclooxygenase-2 Regulates Mesenchymal Cell Differentiation Into the Osteoblast Lineage and is Critically Involved in Bone Repair,” J. Clin. Invest. 109(11):1405-15 (2002), which is hereby incorporated by reference in its entirety). It was found that implantation of an infected transcortical pin always produces lesions adjacent to the pin, and never results in chronic OM in other regions of the tibia or hematogenous spreading in mice (FIGS. 1A-C).

To quantify the osteolysis, a time-course study was performed in which the infected tibiae were analyzed by μCT (FIGS. 1B-C). These results are consistent with sequestrum formation in which osteoclastic bone resorption around the infected implant occurs with concomitant reactive periosteal bone formation.

The presence of OM in the mice was confirmed histologically. FIGS. 2A-H demonstrate that the tibial transcortical pin model contains all of the salient features of chronic OM including: sequestrum and involucrum formation, osteoclastic resorption of the cortical bone and Gram stained extracellular bacteria and biofilm that reside in the necrotic bone surrounding the implant. None of the negative controls, including heat killed S. aureus and non-pathogenic E. coli, demonstrated these features.

Example 2—Real Time PCR Quantitation of Osteomyelitis

There are no known methods to quantify OM. Since it is impossible to effectively extract live bacteria from infected bone to determine bacterial load by classical colony forming units (CFU), a real time PCR method was developed to quantify the number of recoverable nuc genes in DNA samples, as is done to test for contamination in cheese (Hein et al., “Comparison of Different Approaches to Quantify Staphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese,”Appl. Environ. Microbiol. 67(7):3122-6 (2001), which is hereby incorporated by reference in its entirety) and blood (Palomares et al., “Rapid Detection and Identification of Staphylococcus aureus From Blood Culture Specimens Using Real-Time Fluorescence PCR,”Diagn. Microbiol. Infect. Dis. 45(3):183-9 (2003), which is hereby incorporated by reference in its entirety), as a surrogate outcome measure of bacterial load.

RTQ-PCR for the S. aureus-specific nuc gene can be performed using primers 5′-GCGATTGATGGTGATACGGTT-3′ (SEQ ID NO: 15) and 5′-AGCCAAGCCTTGACGAACTAA-3′ (SEQ ID NO: 16) that amplify a previously described 269-bp product (Hein et al., “Comparison of Different Approaches to Quantify Staphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese,”Appl. Environ. Microbiol. 67(7):3122-6 (2001), which is hereby incorporated by reference in its entirety). The reactions can be carried out in a final volume of 20 μl consisting of 0.3 μM primers, 1× Sybr Green PCR Super Mix (BioRad, Hercules, Calif.), and 2 μl of the purified tibia DNA template. The samples can be assayed using a Rotor-Gene RG 3000 (Corbett Research, Sydney, AU).

To control for the integrity of the DNA template between samples, RTQ-PCR can also be performed for the mouse β-actin gene that detects a 124-bp product using primers 5′-AGATGTGAATCAGCAAGCAG-3′ (SEQ ID NO: 17) and 5′-GCGCAAGTTAGGTTTTGTCA-3′ (SEQ ID NO: 18). Using PCR primers specific for murine β-actin, S. aureus nuc, and rRNA genomic DNA, the specificity of these PCRs and the ability to amplify the predicted products was demonstrated (Li et al., “Quantitative Mouse Model of Implant-Associated Osteomyelitis and the Kinetics of Microbial Growth, Osteolysis, and Humoral Immunity,” J. Orthop. Res. 26(1):96-105 (2008), which is hereby incorporated by reference in its entirety). Then, using purified plasmid DNA containing the nuc gene, or S. aureus genomic DNA, a dose response experiment was performed and it was determined that the detection limit for this RTQ-PCR is ˜100 copies (Li et al., “Quantitative Mouse Model of Implant-Associated Osteomyelitis and the Kinetics of Microbial Growth, Osteolysis, and Humoral Immunity,” J. Orthop. Res. 26(1):96-105 (2008), which is hereby incorporated by reference in its entirety). This assay has been used to quantify the in vivo bacterial load as a secondary outcome measure of infection and efficacy of the passive immunization.

Example 3—Kinetics of Infection and Humoral Immunity During the Establishment of Osteomyelitis

To quantify microbial pathogenesis and host immunity during the establishment of osteomyelitis, a time course study was performed in which mice were given an infected transcortical pin implant in their tibia, and the bacterial load and the host humoral response was determined over time by nuc/β-actin RTQ-PCR and western blot, respectively (FIGS. 3A-C). The results indicate a clear inverse correlation between infection and humoral immunity. Consistent with classical microbial pathogenesis and acquired immunity to extracellular bacteria, these results indicate that the bacteria immediately establish themselves and enter an exponential growth phase, which is extinguished by a neutralizing humoral response after 11 days. Based on the coincidence of the peak bacterial load with the genesis of high affinity IgG antibodies against specific bacterial proteins, it is evident that these “immuno-dominant”antigens elicit a functional immune response that is both diagnostic and protective against the establishment of OM.

Example 4—Identification and Cloning of the Glucosaminidase Subunit of S. aureus Autolysin as 56 kDa Immuno-Dominant Antigen that Elicits a Specific IgG2b Response During the Establishment of OM

To further characterize the humoral response during the establishment of OM, the prevalence of Ig isotypes in the serum of mice was determined over the first two weeks of infection by ELISA (Li et al., “Quantitative Mouse Model of Implant-Associated Osteomyelitis and the Kinetics of Microbial Growth, Osteolysis, and Humoral Immunity,”J. Orthop. Res. 26(1):96-105 (2008), which is hereby incorporated by reference in its entirety). The results showed that the mice initiate a classical IgM response in the first week that converts to a specific IgG2b response in the second week, which has recently been shown to have potent opsonic and protective activities against S. aureus antigens (Maira-Litran et al., “Comparative Opsonic and Protective Activities of Staphylococcus aureus Conjugate Vaccines Containing Native or Deacetylated Staphylococcal Poly-N-acetyl-beta-(1-6)-glucosamine,” Infect. Immun. 73(10):6752-62 (2005), which is hereby incorporated by reference in its entirety).

To elucidate the molecular identity of the immuno-dominant antigens identified in FIG. 3C, subtractive Western blotting of total S. aureus extract was performed that was separated by 2D-PAGE (FIGS. 4A-C). This analysis revealed a polypeptide that was not detected by the pre-immune serum, but had strong reactivity with the day 14 post-immune serum. The protein was isolated from a preparative Coomassie blue stained gel, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization (MALDI), which resolved 70 individual peptide peaks. The amino acid sequence from every peptide was a 100% match with the known sequence of the Gmd subunit of S. aureus Alt. Interestingly, others have also recently found Atl to be an immuno-dominant antigen in a rabbit tibia model of MRSA OM (Brady et al., “Identification of Staphylococcus aureus Proteins Recognized by the Antibody-Mediated Immune Response to a Biofilm Infection,” Infect. Immun. 74(6):3415-26 (2006), which is hereby incorporated by reference in its entirety).

To confirm that the spot picked from the 2D-PAGE gel in FIG. 4C was the relevant immuno-dominant antigen, a recombinant 6-His tagged fusion protein was generated by cloning the 1,465 bp coding region of the 53 kDa glucosaminidase subunit of S. aureus autolysin into the Xhol-BamHI site of the pET-28a(+) expression plasmid (Novagen), which contains the lac I promoter for IPTG induction. Following DNA sequencing, the plasmid was used to transform BL21 E. coli, which were used to make recombinant His-glucosaminidase (His-Gmd). This recombinant protein was then used to evaluate the reactivity of pre-immune and immune sera. The results showed that the IPTG induced 57 kDa recombinant protein is only recognized by immune serum, thus confirming that Gmd is a S. aureus immuno-dominant antigen. This experiment was repeated with anti-sera from mice infected with Xen 29, and confirmed that C57Bl/6 also generate Gmd specific antibodies against this bioluminescent strain of S. aureus.

Example 5—In Vivo Bioluminescence Imaging of Lux Transformed S. aureus as a Longitudinal Outcome Measure of OM and Bacterial Growth

Although the RTQ-PCR method of quantifying OM in mouse model is very useful, there are three major limitations to this approach. First, it is not longitudinal, as analysis requires sacrifice of the mice to harvest the DNA. Second, it is very labor intense and requires great care during the DNA isolation, PCR and data analysis. Third, detection of S. aureus genomic DNA (nuc genes) cannot distinguish between bacteria that are in an active growth phase vs. dormant bacteria tightly packed in a biofilm. Thus, RTQ-PCR cannot be readily used to assess mAb effect on bacterial growth in vivo.

To overcome these shortcomings, the present invention relates to a highly innovative approach to monitor pathogens in vivo using bioluminescence imaging (Contag et al., “Photonic Detection of Bacterial Pathogens in Living Hosts,” Mol. Microbiol. 18(4):593-603 (1995), which is hereby incorporated by reference in its entirety). More recently, P. R. Contag and colleagues have generated bioluminescent S. aureus (Xen 29; ATCC 12600) for this purpose (Francis et al., “Monitoring Bioluminescent Staphylococcus aureus Infections in Living Mice Using a Novel luxABCDE Construct,” Infect. Immun. 68(6):3594-600 (2000), which is hereby incorporated by reference in its entirety). FIGS. 5A-B demonstrate how this approach is adapted in the model of OM of the present invention. In a time-course studies with Xen29, only background signal was detected in mice that received a sterile pin or infected mice treated with parenteral gentamycin. In contrast, the BLI of infected, untreated tibiae demonstrated a sharp 4-fold increase from baseline on day 4, which subsequently dropped to background levels by day 11.

Example 6—Recombinant Gmd Vaccine Protects Mice from Implant-Associated OM

To assess the potential of an anti-autolysin passive immunization for OM, an initial active recombinant Gmd vaccine study was performed in which mice (n=20) were immunized as follows: Group 1 (PBS in adjuvant (negative control)); Group 2 (20 μg S. aureus Xen 29 total proteome extract emulsified 1:1 with equal volume of adjuvant (positive control)); Group 3 (20 μg His-glucosaminidase in adjuvant). A 150 μl emulsion of each vaccine was injected intramuscularly (i.m.) 28 day prior to challenge. Booster immunizations (i.m.; 20 μg protein in Freund's incomplete adjuvant) were performed 14 days prior to challenge.

To assess the vaccine efficacy in these mice, an anti-Gmd ELISA was developed (FIG. 6A) and used to quantify serum antibody titers before initial immunization, before booster immunization, and before the bacterial challenge (FIG. 6B). Remarkably, the results demonstrated that only the recombinant vaccine elicited a high titer immune response. To assess the efficacy of these vaccines, the immunized mice were challenged with a Xen29 infected transtibial pin as described in the preceding Example (see FIG. 5A-C), BLI was performed on day 3, and the mice were euthanized for nuc RTQ-PCR on day 11. Remarkably, 18 out of the 20 mice immunized with S. aureus total proteome died within 48 hr of the challenge; thus efficacy data from that group are not available. While only speculative explanations can be provided for this observation (i.e. hyper-immunity to other antigens), the fact that no death occurred in any of the other groups and that the deaths were reproduced in the 4 cohorts of 5 mice in Group 2 indicates that the results are real. For this reason, this immunization protocol should not be used as a positive control for future studies. It also highlights the safety concerns with active vaccines, and supports the rationale of a passive immunization with purified mAb or binding fragments thereof.

The BLI and nuc RTQ-PCR data from Groups 1 and 3 are presented in FIGS. 7A-C. The results clearly demonstrate a significant reduction of BLI detected in the His-Gmd immunized mice (FIGS. 7A-B), which shows a decrease in planktonic growth of the bacteria. Consistent with this finding, it was observed that there was a significant reduction in the number of nuc genes at the peak of the bacterial load in this model (day 11). Thus, these data demonstrate that the recombinant Gmd vaccine can protect mice from OM in the model.

Example 7—Generation and Screening of Mouse Anti-Gmd Monoclonal Antibodies

Based on the success of the His-Gmd immunization described in Example 6, this protocol was used to generate mouse anti-Gmd mAb. Standard procedures were used to generate the mAb. Out of an initial pool of hybridomas that were prepared, a first subset was selected following screened by ELISA for anti-Gmd activity and a second subset possessing higher affinity were selected following a western dot-blot assay.

Five of the hybridoma cell lines were selected based on their apparent high affinity for Gmd (≤10⁻⁹M) and the putative epitope for these regions being found within the R3 domain of Gmd. Because the R3 domain is not the catalytic domain of the Gmd protein, it was unexpected that these monoclonal antibodies would possess as significant anti-Gmd inhibitory activity. The five selected hybridomas were 1C11, 1E12, 2D11, 3A8 and 3H6. All secreted mouse IgG1 antibodies.

Example 8—Alteration of In Vitro Growth of Staphylococcus aureus Xen29 by Monoclonal Anti-glucosaminidase Antibodies

Frozen aliquots of each cell line (1C11, 1E12, 2D11, 3A8 and 3H6) were obtained directly from the vendor who prepared them at our request (Precision Antibodies, Inc., Columbia, Md.). The frozen cells were thawed, and then washed in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 50 μg/mL gentamicin and 10% fetal bovine serum (FBS). Harvested culture supernatant was clarified by centrifugation (10 min, 1000×g) and frozen.

Thirty mL of culture supernatant from each cell line was thawed at 37-45° C. and filtered through a 0.22 μm filter. Each was then purified on a 5 mL bed-volume Protein G-agarose column (GE Healthcare HiTrap™ Protein G HP, Cat. No. 17-0405-03, Lot #10036021). In the place of the pump for which these columns are designed, fluids were added by means of luer-lock syringes fitted by adapters to the top of the column. The column was first washed with PBS to remove the ethanol preservative and then culture supernatants were added at 5-6 mL/min, followed by six column volumes of PBS to wash out unbound protein. Adsorbed antibody was eluted with two column volumes of 0.1 M glycine, pH 2.7, into a collection vessel containing 1 mL of 1.0 M Tris, pH 8.0, to neutralize the eluted product. The column was then washed with four column volumes of PBS in preparation for the next antibody, or with PBS containing 0.02% NaN₃ for storage.

The eluted antibodies were concentrated and dialyzed into PBS in Pierce® concentrators (Thermo Scientific 7 mL/20K MWCO, Cat. No. 87750, lot #KH137631A) by successive centrifugations at 3500×g, 40 min, 4° C. Antibody concentration was determined by ELISA using MOPC21 or Phe12.15 (both mouse IgG1) as standards. Unlabelled goat antimouse IgG (Southern Biotechnology Cat. No. 1030-01) was adsorbed onto 96-well NUNC Maxisorp microtiter plates at 5 μg/mL in PBS, 100 μL per well, one hour RT or overnight at 4° C. Wells were blocked by the addition of 200 μL of 3% BSA in PBS for one hour RT or overnight at 4° C. Blocked plates were washed twice with PBS containing 0.05% Tween 20 (PBST) and ready for use. Samples were prepared as serial dilutions in PBST and 100 μL was added to wells designated for standards and samples. The samples were incubated for one hour, RT, and then washed 4 times with PBST from a squirt bottle. The captured mouse antibody was detected by the addition to each well of 100 μL of HRP-conjugated goat anti-mouse IgG (HRP-GAM; Southern Biotechnology, Cat. No. 1031-05), diluted 1:2000 in PBST, and incubated for one hour, RT. After washing the plates 4 times with PBST from a squirt bottle, the chromogenic HRP substrate ABTS (Southern Biotechnology, Cat. No. 0401-01) was added, 100 μL per well. Color was allowed to develop for 5-10 minutes, RT. Antibody concentrations were determined by projecting sample color values onto the standard curves and then correcting for dilution. These concentrations were used for titration studies to determine their effect on S. aureus growth.

S. aureus Xen29 (Kadurugamuwa et al., “Rapid Direct Method for Monitoring Antibiotics in a Mouse Model of Bacterial Biofilm Infection,” Antimicrob Agents Chemother 47:3130-7 (2003) and Kadurugamuwa et al., “Noninvasive Optical Imaging Method to Evaluate Postantibiotic Effects on Biofilm Infection In Vivo,” Antimicrob Agents Chemother 48:2283-7 (2004), which are hereby incorporated by reference in their entirety) was the only bacterial strain used in these experiments. 1 μL of S. aureus Xen29 was taken from a frozen stock and grown in 10 mL of LB medium at 37° C. on a rotating platform at 200 rpm for 12 hours to mid-log phase. The bacteria were then diluted in LB medium to 1000 cfu/mL and 100 μL of the diluted suspension was added to wells of a flat-bottomed microtiter (with cover) designated for the addition of the antibodies and controls. Each anti-Gmd and control antibody was diluted into LB from stocks about 1 mg/mL in PBS and sterilized through a 0.2μ filter. 100 μL of each antibody was added to designated quadruplicate wells. Plates were then incubated at 37° C. and light scattering was measured at 490 and 670 nm at t=0, 5, 7, 9, 11, 13, 15 hours on a microtiter plate reader. A final time point was taken some time after 24 hours to confirm the measured plateau values.

The five antibodies were purified by affinity chromatography on Protein G-Agarose, concentrated to 1 mg/mL in PBS and dialyzed to remove preservatives such as NaN₃ and antibiotics that might interfere in the assay. 100 cfu of S. aureus strain Xen29 from a mid-log phase culture were placed in each microtiter well in LB medium along with 100 μL of each antibody or control (also in LB medium) at 50 μg/mL (˜3×10⁻⁷ M). Growth was monitored by measuring light scattering at 490 and 670 nm at intervals over 26 hours.

Apparent reduction in the growth of S. aureus was observed by anti-Gmd monoclonal antibodies. As depicted in FIG. 8, four of the antibodies depressed the growth-related increase in light scattering of Xen29. The four antibodies that reduced light scattering all did so to the same degree while the isotype-matched control (MOPC21) was identical to Xen29 grown in the absence of any antibody. The fifth antibody, 3H6, also demonstrated similar behavior in a separate experiment.

It was also observed that the alteration of Xen29 growth-related light scattering is dose-dependent and consistent with high affinity interaction between monoclonal antibodies and native Gmd. The critical decision in addressing dose-dependence of the observed growth alteration of Xen29 was whether the apparent reduction in Xen29 growth was only partially revealed because the concentration of antibody was too low or the maximum effect was already observed and higher concentrations would have no further effect.

Because four of the antibodies had the same magnitude of effect and 50 μg/mL (3×10⁻⁷ M) was a very high concentration, the antibody levels were titrated down from 50 μg/mL in serial 10⁰⁵ dilutions. Representative data is presented in FIG. 9. Monoclonal antibody 1C11 altered the growth of Xen 29 to the same degree at 50, 16 and 5 μg/mL, partially at 1.6 (˜1×10⁻⁸ M), and was not different from the irrelevant antibody MOPC21 at 0.5 ug/mL (FIG. 10). Essentially identical results were obtained for mAbs 2D11, 1E12 and 3A8.

Results for the isotype-matched control antibody MOPC21 are presented in FIG. 10. The growth curves were essentially identical to the no antibody control. The slight elevation of the 50 μg/mL samples was due to their placement in outside wells in the microtiter plate (edge effect).

Assuming that the inhibition of Gmd is due to a high degree of antibody binding, then an estimate assuming that about ten times the K_(a) is required places the operational affinity for the Gmd in the vicinity of 1 nM.

These data indicate that the four anti-Gmd mAbs inhibit Gmd activity, leading to a change in the in vitro growth pattern of Xen29, but they have no effect on the doubling time. The inhibition of Gmd activity leads to failed cytokinesis, i.e., the genome and cell membranes divide normally, but the cell walls fail to separate leading to large clusters of aggregated cells (Sugai et al., “Identification of Endo-beta-N-acetylglucosaminidase and N-acetylmuramyl-L-alanine Amidase as Cluster-dispersing Enzymes in Staphylococcus aureus,” J Bacteriol 177:1491-6 (1995), which is hereby incorporated by reference in its entirety). This change is detectable by light scattering because there are fewer (albeit larger) scattering centers than when the Xen29 cells divide freely.

The antibodies of the present invention have already been shown to satisfy several key criteria. These are high affinity IgG antibodies produced by the parental hybridomas at robust levels (˜50 μg/mL in static culture). They recognize conserved epitopes and consequently some or most will recognize Gmd from the majority of S. aureus strains. In addition, these experiments provide evidence that these monoclonal antibodies recognize native Gmd and not merely the recombinant His-Gmd that was used as the immunogen, and that they act as Gmd inhibitors. The monoclonal antibodies all exhibit Gmd-inhibitory activity of between about 70 to about 80 percent. This level of Gmd-inhibitory activity is rather surprising given that the five selected antibodies bind to epitopes located within the Gmd R3 domain rather than its catalytic domain.

Example 9—Monoclonal Antibodies Specific for Glucosaminidase from Staphylococcus aureus Use a Diverse Array of V_(H) Genes

To determine that the five monoclonal antibodies under investigation were unique, the sequences of the V_(H) and V_(L) genes were determined for the five candidate hybridomas (1C11, 1E12, 2D11, 3A8 and 3H6) identified in the preceding example.

Because there are considerable medical applications and advantages in the study of each of a set of monoclonal antibodies specific for the S. aureus glucosaminidase (Gmd), it is beneficial to identify hybridomas expressing identical antibodies (sibs). Five hybridomas were selected for sequence analysis of their Ig heavy and light chain genes.

Frozen aliquots of the five hybridoma cell lines were obtained from the vendor A&G Precision Antibody™ (9130 Red Branch Road, Suite U, Columbia, Md. 21045), who prepared the hybridomas at our direction. Cells were thawed, washed in DMEM with gentamicin and 10% FBS to remove DMSO, and then cultured in DMEM with gentamicin and 10% FBS. After a few days, cells were harvested by centrifugation and stored at −20° C. as a frozen pellet for subsequent RNA extraction.

RT-PCR amplification of the heavy chain mRNA was successful in the five hybridomas. Sequence analysis revealed that three different germ-line V_(H) gene segments were used. Two of the hybridomas (2D11 and 3H6) used the same germ-line V_(H) and J segments, but different D-segments and each displayed only modest sequence diversification from germ-line at the protein level. The heavy chain from the fifth hybridoma (1C11) did not initially amplify in RT-PCR even with PCR primers designed to amplify some of the more rarely used V_(H) gene-segments. It can be inferred that it expressed an Ig V_(H) gene distinct from the others.

Total RNA was extracted from freshly growing hybridoma cells and ˜5 micrograms of RNA was reverse transcribed using the BioRad iScript kit. Aliquots of cDNA were PCR amplified with consensus primers for the 5′-ends of murine variable heavy and light regions paired with constant region primers. Strong products were obtained for 4/5 V_(H) and 5/5 V_(L) genes. The PCR products were gel purified and directly sequenced. All 4 V_(H) products gave clean sequence, but 2/4 light chain products were mixed. The remaining two gave good sequences. The light chain derived from the cell line that failed to give a heavy chain product was not sequenced. The variable regions for those antibodies that failed to amplify in the initial experiments using framework 1 primers were successfully amplified using a different primer set that targets the secretory leader regions of the variable gene segments. These PCR products were also directly sequenced after purification.

Best matches for germ-line V-region genes were determined by using Ig BLAST (NCBI). The determined DNA sequence was translated into protein sequence by using an on-line program available at Expasy website. Sequence alignment of the 2D11 and 3H6 sequences was performed by visual inspection.

As noted above, eventually all of the five V_(H) genes were successfully amplified and sequenced. Detailed DNA and protein sequence results for the five hybridomas are presented below.

Hybridoma 2D11 (Closest germ line match: J558.18.108) has the V_(H) nucleotide sequence (SEQ ID NO: 21) as follows:

GAGGTGCAGCTGCAGGAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTC AGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTATA TGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTT ATTAATCCTTACAACGGTGATACTACCTACAGCCAGAAGTTCAAGGGCAA GGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCA ACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAAATTAC GACGAGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTC AGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTG CCCAAACTAACTCCATGGTGACCCTGGGATGCCNGGTCAAGGGC

The 2D11 V_(L) (Closest germ line match: at4) has the nucleotide sequence (SEQ ID NO: 22) as follows:

GATATTGTGATGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGA GAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACT GGTACCAGCAGAAGCCAGGATCCTCCCCCAGACTCCTGATTTATGACACA TCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGG GACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAGGATGCTGCCA CTTATTACTGCCAGCAGTGGAGTAGTTACCCGCTCACGTTCGGT

The amino acid sequence of hybridoma 2D11 V_(H) (SEQ ID NO: 1) is as follows:

EVQLQESGPVLVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGV INPYNGDTTYSQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARNY DEYFDVWGTGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCXVKG

The 2D11 V_(L) has the amino acid sequence (SEQ ID NO: 8) as follows:

DIVMTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDT SNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFG

Hybridoma 3H6 (Closest germ line match: J558.18.108) has the V_(H) nucleotide sequence (SEQ ID NO: 23) as follows:

GAGGTGCAGCTGCAGGAGTCTGGACCTGTGCTGGTGAAGCCTGGGGCTTC AGTGAAGCTGTCCTGTAAGGCTTCTGGATACACATTCACTGACTACTTTA TGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGTT ATTAATCCTTTCAACGGTGGTAATAGGTACAACCAGAACTTCAAGGGCAA GGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATGGAGCTCA ACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGGAC TATGACTCCCCCTGGTTTGATTACTGGGGCCAAGGGACTCTGGTCACTGT CTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGAT CTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGC TATTCCCNGAGCCAGTG The 3H6 V_(L) (closest germ line match: cp9, JK1) has the nucleotide sequence (SEQ ID NO: 24) as follows:

CAGATGACACAGACTACGTCCTCCCTGTCTGCCTCTCTGGGAGACAGAGT CACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGT ATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCA AGTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCGGTGGGTCTGGGAC AGATTATTCTCTCTCCATCAGCAACCTGGAACCTGAAGATATTGCCACTT ACTATTGTCAGCAGTATAGTAAGCTTCCTTGGACGTTCGGTGGAGGCACC AAGCTGGAAATCAAA The amino acid sequence of hybridoma 3H6 V_(H) (SEQ ID NO: 2) is as follows:

EVQLQESGPVLVKPGASVKLSCKASGYTFTDYFMNWVKQSHGKSLEWIGV INPFNGGNRYNQNFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARGD YDSPWFDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKG YSXSQ The amino acid sequence of hybridoma 3H6 V_(L) (SEQ ID NO: 9) is as follows:

QMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTS SLHSGVPSRFSGGGSGTDYSLSISNLEPEDIATYYCQQYSKLPWTFGGGT KLEIK

Hybridoma 1E12 (Closest germ line match: 7183.46 VH7) has the V_(H) nucleotide sequence (SEQ ID NO: 25) as follows:

GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTCGTGAAGCCTGGAGGGTC CCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCTATGTCA TGTCTTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACC ATTAGTGATGGTGGTGGTCATACTTACTATCTAGACAATGTAAAGGGCCG ATTCACCATCTCCAGAGACAATGCCAAGAACAACCTGTACCTGCACATGA GCCATCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGAGCTTAC TACGGTAGTAGTTACGACGCTATGGACTACTGGGGTCAAGGAACCTCAGT CACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCC CTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTC AAGGGC The 1E12 V_(L) (Closest germ line match: ai4) has the nucleotide sequence (SEQ ID NO: 26) as follows:

GATATTGTGATCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGA ACGGGTCACCATGACCTGCACTGCCAGCTCAAGTGTAAGTTCCAGTTACT TACACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTNTGGATTTAT AGCACATCCAACCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGG GTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATG CTGCCACTTATTACTGCCACCAGTATCATCGTTCCCCATGGACGTTCGGT GGAGGCACC The amino acid sequence of hybridoma 1E12 V_(H) (SEQ ID NO: 3) is as follows:

EVQLQESGGGFVKPGGSLKLSCAASGFTFSTYVMSWVRQTPEKRLEWVAT ISDGGGHTYYLDNVKGRFTISRDNAKNNLYLHMSHLKSEDTAMYYCARAY YGSSYDAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLV KG The 1E12 V_(L) has the amino acid sequence (SEQ ID NO: 10) as follows:

DIVITQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKXWIY STSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPWTFG GGT

Hybridoma 3A8 (Closest germ line match: VHJ606.4.8.2) has the V_(H) nucleotide sequence (SEQ ID NO: 27) as follows:

GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATC CATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCCTGGA TGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAA ATTAAAGACAAAACTAATAATCATGCAACATACTATGCTGAGTCTGTGAA AGGGAGGTTCACCATCTCAAGAGATGTTTCCAAAAGTCGTGTCTTCCTGC AAATGAACAGCTTAAGACCTGAAGACACTGGCATTTATTACTGTACGTCT GGGCCATATTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTC AGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTG CCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTC CCTGAG The 3A8 V_(L) (closest germ line match: KV 19-25, JK2) has the nucleotide sequence (SEQ ID NO: 28) as follows:

GACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGA CAGGGTCAGCATCACCTGCAAGGCCAGTCAGGACGTGAGTACTGCTGTAG CCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGG ACATCCACCCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATC TGGGACAGATTTTACTCTCACCATCAGCAGTGTGCAGGCTAAAGACCTGG CACTTTATTACTGTCAGCAACATTATACCACTCCGTACACGTTCGGAGGG GGGACCAAGCTGGAAATAAAA The amino acid sequence of hybridoma 3A8 V_(H) (SEQ ID NO: 4) is as follows:

EVQLQESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAE IKDKTNNHATYYAESVKGRFTISRDVSKSRVFLQMNSLRPEDTGIYYCTS GPYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYF PE The amino acid sequence of hybridoma 3A8 V_(L) (SEQ ID NO: 11) is as follows:

DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYW TSTRHTGVPDRFTGSGSGTDFTLTISSVQAKDLALYYCQQHYTTPYTFGG GTKLEIK

Hybridoma 1C11 (closest germline match: VH 9-15, DST4-057B1-6, JH3) has the V_(H) nucleotide sequence (SEQ ID NO: 29) as follows:

CAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGAC AGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAACGTATGGAA TGAGCTGGGTGAATCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGG ATAAACACCTACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGACG GTTTGTCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCA ACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAGAGAGGAG TACAGCTCAGGCTACGCGGCCTGGTTTCCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCTGCA The 1C11 V_(L) (closest germ line match: VK23-43, JK5) has the nucleotide sequence (SEQ ID NO: 30) as follows:

GATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGA TAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTAGCAACAACCTAC ACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCGAATAT GCTTCCCGGTCCATCTCTGGGATCCCCTCTAGGTTCAGTGGCGGTGGATC AGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGTCTGAAGATTTTG GATTGTATTTCTGTCAACAGAGTAACAGCTGGCCGCTCACGTTCGGTGCT GGGACCAAGCTGGAGCTGAAA The amino acid sequence of hybridoma 1C11 V_(H) (SEQ ID NO: 5) is as follows:

QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVNQAPGKGLKWMGW INTYSGVPTYADDFKGRFVFSLETSASTAYLQINNLKNEDTATYFCAREE YSSGYAAWFPYWGQGTLVTVSA The amino acid sequence of hybridoma 1C11 V_(L) (SEQ ID NO: 12) is as follows:

DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIEY ASRSISGIPSRFSGGGSGTDFTLSINSVESEDFGLYFCQQSNSWPLTFGA GTKLELK

From the obtained sequences the closest fit for germ line V_(H) and V_(L) gene segments was determined as shown in Table 1.

TABLE 1 Germline Matches for Sequenced Hybridoma Cell Lines Hybridoma Germ Line V_(H) Germ Line V_(L) 1C11 VH 9-15, DST4-C57Bl-6, JH3 VK23-43, JK5 1E12 7183.46 VH7 ai4 2D11 J558.18.108 at4 3A8 VHJ606.48.2 KV 19-25, JK2 3H6 J558.18.108 cp9, JK1

Each of the five hybridomas expressed a unique V_(H) gene. FIG. 11 shows the alignment of the four initially sequenced V_(H) genes, and FIG. 13 shows the alignment of all five sequenced V_(H) genes. FIG. 15 shows the alignment of the sequenced V_(H) genes for 1C11 and 3A8 only.

FIG. 12 shows the alignment of the two initially obtained V_(L) genes. FIG. 14A shows the alignment of all five sequenced V_(I) genes, whereas FIG. 14B all five sequenced V_(I) genes except of that for 1C11.

The fact that one hybridoma (1C11) did not yield a PCR product with the initial primers that amplify the most common V_(H) genes suggests that it uses a fourth V_(H) gene segment not identical to any of the others identified here. Two of the hybridomas used the same V_(H) germ line gene, but are not identical. Hybridomas 2D11 and 3H6 both used the germ line V_(H) gene segment J558.18.108. Their sequences are compared with the germ line gene in FIG. 11. Inspection of the sequence reveals that there is only one difference in CDR1, four in CDR2 and five in CDR3 (including gaps).

The five hybridomas are not sibs and among them at least four V_(H) germ line genes were utilized.

Example 10—Inhibition of S. aureus Gmd Enzymatic Activity by mAbs 1C11, 2D11, 3H6, 1E12, and 3A8

The method of measurement of Gmd enzymatic activity is essentially the method of Mani et al. (Mani et al., “Isolation and Characterization of Autolysis-Defective Mutants of Staphylococcus aureus Created by Tn917-lacZ Mutagenesis” J. Bacteriol. 175(5): 1493-1499 (1993)). Lyophilized and resuspended Micrococcus lysodeikticus were degraded by the action of the Gmd resulting in a reduction in light scattering at 490 nm. 100 μL of sample containing Gmd diluted in phosphate-buffered saline with 0.05% Tween 20 (PBST) was added to the wells of a 96-well microtiter plate. 100 μL of a 0.15% (w/v) suspension of Micrococcus lysodeikticus was added to each well and the light scattering was measured immediately to establish the initial A₄₉₀, typically about 0.8. The plate was then incubated at 37° C. and light scattering was re-measured at 30 and 60 minutes. Reduction in A₄₉₀ at 60 minutes was taken as the measure of Gmd activity. The modest background rate (no Gmd) was subtracted. This method does not distinguish Gmd activity from lysozyme activity.

To measure inhibition of Gmd enzymatic activity the Gmd was pre-titered to determine the concentration that will yield about a 50% reduction in A₄₉₀ in 60 minutes. Then, 50 μL of antibody diluted in PBST was added to each well of a 96-well microtiter plate followed by 50 μL of appropriately diluted Gmd, and the mixtures were allowed to incubate for 5 or more minutes, and finally 100 μL of 0.15% Micrococcus lysodeikticus was added and the initial A₄₉₀ was measured. The plate was then incubated at 37° C. and the A₄₉₀ was measured at 30 and 60 minutes. Percent inhibition was calculated as 100*(1−(Δ₆₀A₄₉₀ inhibitor/Δ₆₀A₄₉₀ no inhibitor control)).

In FIG. 18, serial dilutions of each of the five antibodies plus the isotype-matched negative control MOPC21 were assessed for their ability to inhibit the catalytic activity of recombinant S. aureus His-Gmd (Gmd) or hen egg lysozyme (HEL). Each of the five anti-Gmd monoclonal antibodies inhibited His-Gmd activity by 75-80%, while displaying no inhibitory activity with HEL. MOPC21 had no inhibitory activity with either enzyme. The degree of inhibition of His-Gmd has not been observed to exceed 80% by these five antibodies. High, though partial, inhibition is one of the characteristics of this group of antibodies.

The ability of the five antibodies to inhibit the native Gmd enzyme secreted by S. aureus strain UAMS-1 is depicted in FIG. 19. As with the recombinant Gmd, the native Gmd was inhibited about 80% by each antibody. By the measure of enzyme inhibition, the antibodies react similarly with both the native and recombinant Gmds.

An SEM analysis of anti-Gmd activity for several monoclonal antibodies is shown in FIGS. 20C-D. Xen29 S. aureus was grown for 12 hours in Luria-Bertani broth to achieve a mid-log growth suspension, and then 10,000 CFU were incubated with: (FIGS. 20A-B) no antibody, (FIG. 20C) 50 μg/ml 1E12, or (FIG. 20D) 50 μg/ml 1C11 for 1 hour. Samples were then plated onto sterile silicon chips, fixed, dehydrated, and coated with gold for visualization by scanning electron microscopy. Micrographs (FIGS. 20C-D) illustrate the effect of anti-Gmd antibody, promoting formation of large clusters and cell-independent lysis (arrows) of approximately 20% of the cells.

Example 11—Passive Vaccine Containing Anti-Gmd mAbs Inhibits Staphylococcus aureus In Vivo Following Orthopedic Implant in Mouse OM Model

The OM model with trans-tibial pin (see Examples 1 and 6) was used to assess the ability of candidate mAbs 1C11 and 3A8 to inhibit S. aureus growth in vivo. Briefly, five week old female BALB/cJ mice received an intraperitoneal injection of saline (n=10) or 1 mg of purified 3A8 anti-Gmd antibody (n=5) in 0.25 ml saline or 31C11 A8 anti-Gmd antibody (n=5) in 0.25 ml saline 3 days prior to surgery. At surgery, the mice received a transtibial implant containing 500,000 CFU of Xen29 S. aureus. The mice were imaged to assess bioluminescence on days 0, 3, 5, 7, 10 or 11, and 14, and images with the BLI heat map from a representative animal in each group are shown in FIGS. 21A and 22A. Of note is the absence of a BLI signal in the anti-Gmd 3A8 animal until day 11 and 1C11 animal until day 5, presumably when the antibody titer decreased below the effective concentration. The BLI values on day 3 for each mouse in the study are shown with the mean for each group (FIG. 21B, p=0.02; FIG. 22B). For 1C11, it is interesting to note that this therapy cured 50% of the animals at day 3. X-rays from a representative animal in each group obtained on day 14 is shown to illustrate the osteolytic lesion (arrow) in the placebo mouse, which was not present in the anti-Gmd treated animals (FIGS. 21C, 22C).

Example 12—Generation of Humanized Antibody

The variable regions of the light and heavy chains of the 1C11 antibody were re-amplified from the purified hybridoma PCR product described in Example 9 using primers to permit cloning into the human antibody expression vectors described by Tiller et al. (“Efficient Generation of Monoclonal Antibodies from Single Human B Cells by Single Cell RT-PCR and Expression Vector Cloning,” J. Immunol. Methods 329(1-2):112-24 (2008), which is hereby incorporated by reference in its entirety). Plasmids containing the 1C11 light and heavy chain variable regions and human kappa and IgG1 constant regions were prepared and co-transfected into HEK293 cells. After 3 days, the medium was removed from the cells and assayed for the presence of human IgG and for binding to immobilized Gmd protein by ELISA. Bound antibody was detected using a goat anti-Human IgG antibody coupled to horseradish peroxidase and 3,3′, 5, 5′ tetramethylbenzidene substrate.

To establish that the human:mouse chimeric 1C11 (h1C11) reacted with Gmd as well as the parental mouse 1C11, each was tested for its ability to inhibit the enzymatic activity of His-Gmd. Both h1C11 and mouse 1C11 displayed nearly identical inhibitory activity (FIG. 23), thereby demonstrating that the chimeric IgG molecule retained the binding activity of the parent.

A similar procedure will be performed using the human CDR1 and CDR2 homologs of 1C11 identified in FIGS. 17A-B, and a CDR3 region from one or more candidate D regions including, without limitation, IGHD5-5, 18, and 12*01.

The humanized 1C11 antibody and antibody comprising the human CDR1 and CDR2 homologs of 1C11 can be utilized in a phase I clinical trial in elderly patients (>65 yrs) undergoing primary total joint replacement.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. 

What is claimed:
 1. A monoclonal antibody or binding portion thereof that binds specifically to a Staphylococcus aureus glucosaminidase and inhibits in vivo growth of S. aureus. 